U.S. patent application number 10/267811 was filed with the patent office on 2003-06-12 for methods of using 279, a human g protein-coupled protein receptor.
This patent application is currently assigned to Millennium Pharmaceuticals, Inc.. Invention is credited to Galvin, Katherine M., Logan, Thomas Joseph.
Application Number | 20030109044 10/267811 |
Document ID | / |
Family ID | 23286382 |
Filed Date | 2003-06-12 |
United States Patent
Application |
20030109044 |
Kind Code |
A1 |
Logan, Thomas Joseph ; et
al. |
June 12, 2003 |
Methods of using 279, a human G protein-coupled protein
receptor
Abstract
The invention provides isolated nucleic acids molecules,
designated 279 nucleic acid molecules, which encode human G
protein-coupled receptor (GPCR) family members. The invention also
provides antisense nucleic acid molecules, recombinant expression
vectors containing 279 nucleic acid molecules, host cells into
which the expression vectors have been introduced, and nonhuman
transgenic animals in which a 279 gene has been introduced or
disrupted. The invention still further provides isolated 279
proteins, fusion proteins, antigenic peptides and anti-279
antibodies. Methods utilizing compositions of the invention to
treat, prevent or diagnose angiogenic disorders, e.g.,
cardiovascular and cancerous disorders, are also provided.
Inventors: |
Logan, Thomas Joseph;
(Needham, MA) ; Galvin, Katherine M.; (Jamaica
Plain, MA) |
Correspondence
Address: |
MILLENNIUM PHARMACEUTICALS, INC.
75 Sidney Street
Cambridge
MA
02139
US
|
Assignee: |
Millennium Pharmaceuticals,
Inc.
|
Family ID: |
23286382 |
Appl. No.: |
10/267811 |
Filed: |
October 9, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60329648 |
Oct 16, 2001 |
|
|
|
Current U.S.
Class: |
435/455 ;
424/155.1; 514/1.9; 514/13.3; 514/16.4; 514/19.3; 514/20.6;
514/44R |
Current CPC
Class: |
C07K 14/705 20130101;
C07K 14/723 20130101; A61K 38/00 20130101 |
Class at
Publication: |
435/455 ; 514/12;
514/44; 514/7; 424/155.1 |
International
Class: |
A61K 039/395; A61K
038/17; A61K 038/16; A61K 048/00; C12N 015/85 |
Claims
What is claimed is:
1. A method of modulating the activity of a 279-expressing
endothelial cell, comprising contacting the cell with a compound
that modulates the activity or expression of a 279 polypeptide or
nucleic acid, in an amount effective to modulate the activity of
the cell.
2. The method of claim 1, wherein the compound is selected from the
group consisting of a peptide, a phosphopeptide, a small organic
molecule, a small inorganic molecule and an antibody.
3. A method of treating or preventing an angiogenic disorder, in a
subject, comprising administering to the subject a compound that
modulates the activity or expression of a 279 polypeptide or
nucleic acid, in an amount effective to treat or prevent said
cardiovascular disorder.
4. The method of claim 3, wherein the angiogenic disorder involves
pathological angiogenesis or abnormal neovascularization.
5. The method of claim 3, wherein the angiogenic disorder is
atherosclerosis.
6. The method of claim 3, wherein the angiogenic disorder is a
cancer.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. provisional
application No. 60/329,648, filed Oct. 16, 2001, the contents of
which are incorporated by reference herein.
BACKGROUND OF THE INVENTION
[0002] G-protein coupled receptors (GPCRs) are seven transmembrane
domain proteins that mediate signal transduction of a diverse
number of ligands through heterotrimeric G proteins (Strader, C. D.
et al. (1994) Annu. Rev. Biochem. 63: 101-132). G protein-coupled
receptors (GPCRs), along with G-proteins and effector proteins
(e.g., intracellular enzymes and channels), are the components of a
modular signaling system. Upon ligand binding to an extracellular
portion of a GPCR, different G proteins are activated, which in
turn modulate the activity of different intracellular effector
enzymes and ion channels (Gutkind, J. S. (1998) J. Biol. Chem. 273:
1839-1842; Selbie, L. A. and Eill, S. J. (1998) Trends Pharmacol.
Sci. 19:87-93).
[0003] G proteins represent a family of heterotrimeric proteins
composed of a, b and g subunits, which bind guanine nucleotides.
These proteins are usually linked to cell surface receptors (e.g.,
a GPCR). Following ligand binding to a GPCR, a conformational
change is transmitted to the G protein, which causes the
.alpha.-subunit to exchange a bound GDP molecule for a GTP molecule
and to dissociate from the .beta..gamma.-subunits. The GTP-bound
form of the a-subunit typically functions as an effector-modulating
moiety, leading to the production of second messengers, such as
cyclic AMP (e.g., by activation of adenylate cyclase),
diacylglycerol or inositol phosphates. Greater than 20 different
types of a-subunits are known in man, which associate with a
smaller pool of b and g subunits. Examples of mammalian G proteins
include Gi, Go, Gq, Gs and Gt (Lodish H. et al. Molecular Cell
Biology, (Scientific American Books Inc., New York, N.Y.,
1995).
[0004] One subfamily of seven transmembrane receptors is the
rhodopsin family. Proteins of this family can be expressed in
photoreceptor cells. They generally contain a prosthetic group,
11-cis-retinal. Absorption of light by retinal causes an
isomerization in the molecule and consequently a conformational
change in the rhodopsin protein. This structural change is
transmitted to a signaling cascade by means of the coupled G
protein. While their activating ligands vary, the amino acid
sequences are very similar ad are believed to adopt a common
structural framework comprising seven transmembrane TM helices.
[0005] The increased number of GPCR proteins in endothelial cells
and smooth muscle cells provides and opportunity to pursue their
role in cardiovascular and angiogenesis related disorders including
atherosclerosis and cancer. Identification of new methods of using
GPCR proteins is an important step in treating these disorders,
including those disorders in which acceptable treatment has yet to
be discovered.
SUMMARY OF THE INVENTION
[0006] Nucleotide and corresponding amino acid sequences for a G
protein coupled receptor (GPCR), referred to herein as "279" are
disclosed. The nucleotide sequence of a cDNA encoding 279 is shown
in SEQ ID NO: 1, and the amino acid sequence of a 279 polypeptide
is shown in SEQ ID NO: 2. In addition, the nucleotide sequences of
the coding region are depicted in SEQ ID NO: 3. 279 protein is
homologous to G protein-coupled receptors, and in particular human
GPR4 (Accession No. L36148; Heiber, M. et al. (1995) DNA Cell Biol.
14(1), 25-35). Applicants have shown expression of 279 mRNA in
human and rodent endothelial tissues, such as heart and blood
vessels. Accordingly, modulators of 279 polypeptide activity or
expression may be used to treat or prevent angiogenic disorders,
such as cardiovascular and cancerous disorders.
[0007] Accordingly, in one aspect, the invention features a nucleic
acid molecule that encodes a 279 protein or polypeptide, e.g., a
biologically active portion of the 279 protein. In a preferred
embodiment the isolated nucleic acid molecule encodes a polypeptide
having the amino acid sequence of SEQ ID NO: 2. In other
embodiments, the invention provides isolated 279 nucleic acid
molecules having the nucleotide sequence shown in SEQ ID NO: 1, SEQ
ID NO: 3. In still other embodiments, the invention provides
nucleic acid molecules that are substantially identical (e.g.,
naturally occurring allelic variants) to the nucleotide sequence
shown in SEQ ID NO: 1, SEQ ID NO: 3. In other embodiments, the
invention provides a nucleic acid molecule which hybridizes under a
stringency condition described herein to a nucleic acid molecule
comprising the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3,
wherein the nucleic acid encodes a full length 279 protein or an
active fragment thereof.
[0008] In a related aspect, the invention further provides nucleic
acid constructs that include a 279 nucleic acid molecule described
herein. In certain embodiments, the nucleic acid molecules of the
invention are operatively linked to native or heterologous
regulatory sequences. Also included, are vectors and host cells
containing the 279 nucleic acid molecules of the invention e.g.,
vectors and host cells suitable for producing 279 nucleic acid
molecules and polypeptides.
[0009] In another related aspect, the invention provides nucleic
acid fragments suitable as primers or hybridization probes for the
detection of 279-encoding nucleic acids.
[0010] In still another related aspect, isolated nucleic acid
molecules that are antisense to a 279 encoding nucleic acid
molecule are provided.
[0011] In another aspect, the invention features, 279 polypeptides,
and biologically active or antigenic fragments thereof that are
useful, e.g., as reagents or targets in assays applicable to
treatment and diagnosis of 279-mediated or -related disorders. In
another embodiment, the invention provides 279 polypeptides having
a 279 activity. Preferred polypeptides are 279 proteins including
at least one GPCR domain, seven hydrophobic membrane-spanning
regions, three extracellular loops alternate with three
intracellular loops, and, preferably, having a 279 activity, e.g.,
a 279 activity as described herein.
[0012] In other embodiments, the invention provides 279
polypeptides, e.g., a 279 polypeptide having the amino acid
sequence shown in SEQ ID NO: 2; an amino acid sequence that is
substantially identical to the amino acid sequence shown in SEQ ID
NO: 2; or an amino acid sequence encoded by a nucleic acid molecule
having a nucleotide sequence which hybridizes under a stringency
condition described herein to a nucleic acid molecule comprising
the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, wherein the
nucleic acid encodes a full length 279 protein or an active
fragment thereof.
[0013] In a related aspect, the invention further provides nucleic
acid constructs that include a 279 nucleic acid molecule described
herein.
[0014] In a related aspect, the invention provides 279 polypeptides
or fragments operatively linked to non-279 polypeptides to form
fusion proteins.
[0015] In another aspect, the invention features antibodies and
antigen-binding fragments thereof, that react with, or more
preferably specifically bind 279 polypeptides or fragments thereof,
e.g., a seven-transmembrane receptor domain, an extracellular loop,
an intracellular loop, or an N- or C-terminal domain, of a 279
polypeptide.
[0016] In another aspect, the invention provides methods of
screening for compounds that modulate the expression or activity of
the 279 polypeptides or nucleic acids.
[0017] In still another aspect, the invention provides a process
for modulating 279 polypeptide or nucleic acid expression or
activity, e.g. using the screened compounds. Examples of 279
activities include modulation of proliferation, survival, migration
and tube formation of a cell, e.g., an endothelial cell; increased
nitric oxide (NO) production; and modulation of vascular tone.
[0018] In certain embodiments, the methods involve treatment of
conditions related to aberrant activity or expression of the 279
polypeptides or nucleic acids, such as conditions or disorders
involving aberrant or deficient endothelial cell activity, e.g.,
aberrant or deficient endothelial cell proliferation,
differentiation, survival, or migration; or other cardiovascular
conditions or disorders, e.g., blood vessel associated disorders
(e.g., atherosclerosis); aberrant or deficient angiogenesis, e.g.,
angiogenic disorders; aberrant or deficient cellular proliferation
or differentiation, e.g., cancers (e.g., cancers of the neural
organs (e.g., neuroblastomas or glioblastomas), hemangiomas or a
Wilm's tumor); or neurological disorders.
[0019] In yet another aspect, the invention provides methods for
modulating the activity of a 279-expressing cell. The method
includes contacting the cell with an agent, e.g., compound (e.g., a
compound identified using the methods described herein) that
modulates the activity, or expression, of the 279 polypeptide or
nucleic acid. In a preferred embodiment, the contacting step is
effective in vitro or ex vivo. In other embodiments, the contacting
step is effected in vivo, e.g., in a subject (e.g., a mammal, e.g.,
a human), as part of a therapeutic or prophylactic protocol.
[0020] Activities of a 279-expressing cell include modulation of
proliferation, survival, migration and capillary tube formation of
a cell, e.g., an endothelial cell; increased nitric oxide (NO)
production; and modulation of vascular tone, e.g., vasoconstriction
or vasodilation.
[0021] In one embodiment, the activity of the 279-expressing cell
is inhibited. For example, the angiogenic activity of the
279-expressing cell can be inhibited using the agents described
herein to thereby treat or prevent disorders involving aberrant
angiogenesis (also referred to herein as "angiogenic disorders"),
e.g., cancer or tumor growth, or atherosclerosis.
[0022] In other embodiments, the activity of the 279-expressing
cell is enhanced. For example, the angiogenic activity of the
279-expressing cell can be enhanced using the agents described
herein to thereby treat or prevent disorders involving aberrant
angiogenesis, e.g., cardiovascular disorders.
[0023] In a preferred embodiment, the 279-expressing cell or tissue
is an endothelial cell or tissue, or a smooth muscle cell or
tissue. Therefore, the methods of the invention can be used to
treat, prevent and/or diagnose an angiogenic or an endothelial
cell-mediated disorder, e.g., a disorder involving aberrant
proliferation, migration, angiogenesis, or vascularization.
Examples of angiogenic or endothelial cell disorders include
tumorigenesis, tumor metastasis, psoriasis, diabetic retinopathy,
endometriosis, Grave's disease, ischemic disease, atherosclerosis,
and chronic inflammatory diseases (e.g., rheumatoid arthritis). In
other embodiments, the 279-expressing cell is a heart cell, a liver
cell, a blood vessel-associated cell, or a neural cell, e.g., a
brain or a glial cell. In other embodiments, the 279-expressing
cell is a cancer cell, e.g., a cell from a solid or a soft tissue
tumor, or a metastatic cell.
[0024] In a preferred embodiment, the subject is a human suffering
from, or at risk of, a 279-mediated disorder or disease, e.g., an
angiogenic disorder, e.g., a cardiovascular disorder (e.g.,
atherosclerosis, for example, a human with early, intermediate or
advanced atherosclerosis), an endothelial cell disorder, or a
cancer, as described herein. For example, the subject is a patient
undergoing a therapeutic or prophylactic protocol. In other
embodiments, the subject is a non-human animal, e.g., an
experimental animal.
[0025] In those embodiments where inhibition of 279 activity is
desired (e.g., treatment or prevention of cancers), the agent,
e.g., the compound, is an inhibitor of a 279 polypeptide.
Preferably, the agent, e.g., the inhibitor is chosen from a
peptide, a phosphopeptide, a small organic molecule, a small
inorganic molecule and an antibody (e.g., an antibody conjugated to
a therapeutic moiety selected from a cytotoxin, a cytotoxic agent
and a radioactive metal ion). In other embodiments, the inhibitor
inhibits angiogenesis. For example, the inhibitor is a
carboxyamido-triazole. which inhibits angiogenesis by blocking the
calcium-mediated nitric-oxide synthase-vascular endothelial growth
factor pathway. In another preferred embodiment, the agent, e.g.,
compound, is an inhibitor of a 279 nucleic acid, e.g., an
antisense, a ribozyme, or a triple helix molecule.
[0026] In those embodiments where activation of 279 activity is
desired (e.g., treatment or prevention of cardiovascular disorder),
the agent, e.g., compound, is an activator or an agonist of a 279
polypeptide. Examples of agonists of 279 activity include 279
ligands, e.g., a phospholipid (e.g., sphingolipids, e.g.,
sphingosylphosphorylcholine, or lysophosphatidylcholine).
[0027] The agent(s) described herein can be administered by
themselves, or in combination with at least one more agent
(referred to herein as a "second agent(s)"), or procedures.
Examples of second agents include cytotoxic agents, such as
anti-microtubule agent, a topoisomerase I inhibitor, a
topoisomerase II inhibitor, an anti-metabolite, a mitotic
inhibitor, an alkylating agent, an intercalating agent, an agent
capable of interfering with a signal transduction pathway, an agent
that promotes apoptosis or necrosis, and radiation. In other
embodiments, the second agent is a cholesterol-lowering agent. In
other embodiments, the agent(s) of the invention is administered in
combination with an interventional procedure ("procedural vascular
trauma").
[0028] In other embodiments, the second agent is an activator or an
agonist of a 279 polypeptide. Examples of agonists of 279 activity
include 297 ligands, e.g., a phospholipid (e.g.,
sphingosylphosphorylchol- ine or lysophosphatidylcholine).
[0029] The second agent or procedure can be administered or
effected prior to, at the same time, or after administration of the
agent(s) of the invention, in single or multiple administration
schedules. For example, the second agent and the agents of the
invention can be administered continually over a preselected period
of time, or administered in a series of spaced doses, i.e.,
intermittently, for a period of time.
[0030] In a preferred embodiment, the method further includes
removing from the subject 279-expressing cells (e.g., endothelial
cells or smooth muscle cells), e.g., by separating the
279-expressing cells from the other cells.
[0031] In another aspect, the invention features methods for
treating or preventing a disorder characterized by aberrant
activity, e.g., cellular proliferation, differentiation, or
migration, of a 279-expressing cell or tissue, in a subject.
Preferably, the method includes administering to the subject (e.g.,
a mammal, e.g., a human) an effective amount of an agent, e.g., a
compound (e.g., a compound identified using the methods described
herein) that modulates the activity, or expression, of the 279
polypeptide or nucleic acid, thereby treating or preventing the
disorder.
[0032] In a preferred embodiment, the 279-expressing cell or tissue
is an endothelial cell or tissue, or a smooth muscle cell or
tissue. Therefore, the methods of the invention can be used to
treat, prevent and/or diagnose an angiogenic or an endothelial
cell-mediated disorder, e.g., a disorder involving aberrant
proliferation, migration, angiogenesis, or vascularization. In one
embodiment, the 279-expressing cell is a cancer cell, e.g., a cell
from a solid or a soft tissue tumor, or a metastatic cell. In other
embodiments, the 279-expressing cell is a heart cell, a blood
vessel-associated cell, a liver cell, or a neural cell, e.g., a
brain or a glial cell.
[0033] In a preferred embodiment, the disorder is a cardiovascular
disorder, e.g., a blood vessel associated disorder (e.g.,
atherosclerosis, for example, a human with early, intermediate or
advanced atherosclerosis); an angiogenic or an endothelial cell
disorder, e.g., a disorder involving aberrant proliferation,
migration, angiogenesis, or vascularization. In other embodiments,
the disorder is a cancer, a liver disorder, or a neurological
disorder.
[0034] Examples of cardiovascular disorders that can be treated or
prevented with the methods of the invention include, but are not
limited to, atherosclerosis, myocardial infarction, stroke,
thrombosis, aneurism, heart failure, ischemic heart disease, angina
pectoris, sudden cardiac death, hypertensive heart disease;
non-coronary vessel disease, such as arteriolosclerosis, small
vessel disease, nephropathy, hypertriglyceridemia,
hypercholesterolemia, hyperlipidemia, xanthomatosis, asthma,
hypertension, emphysema and chronic pulmonary disease; or a
cardiovascular condition associated with interventional procedures
("procedural vascular trauma"), such as restenosis following
angioplasty, placement of a shunt, stent, synthetic or natural
excision grafts, indwelling catheter, valve or other implantable
devices. Preferred cardiovascular disorders include
atherosclerosis, myocardial infarction, aneurism, and stroke.
[0035] Examples of angiogenic or endothelial cell disorders include
tumorigenesis, tumor metastasis, psoriasis, diabetic retinopathy,
endometriosis, Grave's disease, ischemic disease (e.g.,
atherosclerosis), arterial-venular malformations (e.g.,
hemangiomas), and chronic inflammatory diseases (e.g., rheumatoid
arthritis).
[0036] Examples of cancerous disorders include solid tumors (e.g.,
carcinoma, sarcoma); soft tissue tumors, e.g., hematopoietic
neoplastic disorders, e.g., leukemias; or metastatic disorders. A
metastatic tumor can arise from a multitude of primary tumor types,
including but not limited to those of prostate, colon, lung, breast
and liver origin.
[0037] In a preferred embodiment, the subject is a human suffering
from, or at risk of, a 279-mediated disorder or disease, e.g., a
cardiovascular disorder (e.g., atherosclerosis, for example, a
human with early, intermediate or advanced atherosclerosis), or an
endothelial cell disorder, a cancer, as described herein.
[0038] In a further aspect, the invention provides methods for
evaluating the efficacy of a treatment of a disorder, e.g., a
cardiovascular, endothelial or proliferative disorder. The method
includes: treating a subject, e.g., a patient or an animal, with a
protocol under evaluation (e.g., treating a subject with one or
more of: chemotherapy, radiation, and/or an agent, e.g., a
compound, identified using the methods described herein or a second
agent or procedure as described herein); and evaluating the
expression of a 279 nucleic acid or polypeptide before and after
treatment. A change, e.g., a decrease or increase, in the level of
a 279 nucleic acid (e.g., mRNA) or polypeptide after treatment,
relative to the level of expression before treatment, is indicative
of the efficacy of the treatment of the disorder. The level of 279
nucleic acid or polypeptide expression can be detected by any
method described herein.
[0039] In a preferred embodiment, the evaluating step includes
obtaining a sample (e.g., a tissue sample, e.g., a biopsy, or a
fluid sample) from the subject, before and after treatment and
comparing the level of expressing of a 279 nucleic acid (e.g.,
mRNA) or polypeptide before and after treatment.
[0040] In another aspect, the invention provides methods for
evaluating the efficacy of a therapeutic or prophylactic agent
(e.g., a second agent or procedure as described herein, or an
anti-neoplastic agent). The method includes: contacting a sample
with an agent (e.g., a compound identified using the methods
described herein, a cytotoxic agent) and, evaluating the expression
of 279 nucleic acid or polypeptide in the sample before and after
the contacting step. A change, e.g., a decrease or increase, in the
level of 279 nucleic acid (e.g., mRNA) or polypeptide in the sample
obtained after the contacting step, relative to the level of
expression in the sample before the contacting step, is indicative
of the efficacy of the agent. The level of 279 nucleic acid or
polypeptide expression can be detected by any method described
herein. In a preferred embodiment, the sample includes cells
obtained from a cardiovascular or blood vessel-associated tissue;
an endothelial tissue; or a cancerous tissue.
[0041] The invention also provides assays for determining the
activity of or the presence or absence of 279 polypeptides or
nucleic acid molecules in a biological sample, including for
disease diagnosis.
[0042] In further aspect, the invention provides assays for
determining the presence or absence of a genetic alteration in a
279 polypeptide or nucleic acid molecule, including for disease
diagnosis.
[0043] In another aspect, the invention features a two dimensional
array having a plurality of addresses, each address of the
plurality being positionally distinguishable from each other
address of the plurality, and each address of the plurality having
a unique capture probe, e.g., a nucleic acid or peptide sequence.
At least one address of the plurality has a capture probe that
recognizes a 279 molecule. In one embodiment, the capture probe is
a nucleic acid, e.g., a probe complementary to a 279 nucleic acid
sequence. In another embodiment, the capture probe is a
polypeptide, e.g., an antibody specific for 279 polypeptides. Also
featured is a method of analyzing a sample by contacting the sample
to the aforementioned array and detecting binding of the sample to
the array.
[0044] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0045] FIG. 1 depicts a hydropathy plot of human 279. Relative
hydrophobic residues are shown above the dashed horizontal line,
and relative hydrophilic residues are below the dashed horizontal
line. Numbers corresponding to positions in the amino acid sequence
of human 279 are indicated. Polypeptides of the invention include
fragments which include: all or part of a hydrophobic sequence,
i.e., a sequence above the dashed line, e.g., the sequence from
about amino acid 23 to 43, from about 52 to 74, from about 92 to
113, from about 134 to 154, from about 186 to 205, from about 225
to 245, and from about 272 to 289 of SEQ ID NO: 2; all or part of a
hydrophilic sequence, i.e., a sequence below the dashed line, e.g.,
the sequence from about amino acid 77 to 91, from about 156 to 170,
and from about 207 to 224 of SEQ ID NO: 2.
[0046] FIG. 2 depicts an alignment of the GPCR domain of human 279
with a consensus amino acid sequence derived from a hidden Markov
model (HMM) from PFAM. The upper sequence is the consensus amino
acid sequence (SEQ ID NO: 4), while the lower amino acid sequence
corresponds to amino acids 34 to 286 of SEQ ID NO: 2.
[0047] FIG. 3 is a bar graph depicting increased expression of
murine 279 mRNA in the developing mouse heart. In rodents, there is
major proliferation and development of the cardiac blood vessels
during the first three weeks after birth (Tomanek, R J (1996)
Cardiovasc Res. 31: E46-E51, and Olivetti, G et al. (1980) Circ.
Res. 46: 503-512). FIG. 3 is a panel that includes pooled heart
samples from several timepoints during the period of vascular
growth (5, 7, 10, 14, 17, 21 days after birth), as well as the
mothers of these newborns ("M", growth state of vessels in heart is
not well-defined) and normal adults ("A"), with quiescent
vasculature. Regulation during the newborn period, or differential
expression between newborns and adults supports an angiogenic
function for 279 protein.
[0048] FIG. 4 depicts expression of m279 mRNA in a superovulation
model. Expression of m279 mRNA in this model parallels that of
endothelial and angiogensis markers, such as vascular endothelial
growth factor (VEGF), consistent with a role of 279 polypeptide in
angiogenesis.
[0049] FIG. 5 is a bar graph depicting expression/regulation of 279
mRNA in endothelial cells (ECs) isolated from the indicated tissues
and exposed to the indicated treatments. Expression of 279 mRNA is
upregulated in proliferating endothelial cells compared to
confluent endothelial cells.
[0050] FIG. 6 is a bar graph depicting 279 gene regulation in ECs
in response to starvation and angiogenic growth factor
stimulation.
[0051] FIG. 7 is a bar graph depicting decrease expression of 279
mRNA as cells become confluent (70% vs. 90%) or with starvation
(70% and/or 90% vs. 18 h starve), and upregulation of the mRNA at 2
h or 18 h in response to both growth factors (CM) and/or a single
growth factor. Id1 is used as a control gene.
DETAILED DESCRIPTION
[0052] The human 279 sequence (see SEQ ID NO: 1, as recited in
Example 1), which is approximately 1089 nucleotides long including
untranslated regions, contains a predicted methionine-initiated
coding sequence of about 1089 nucleotides, including the
termination codon. The coding sequence encodes a 362 amino acid
protein (see SEQ ID NO: 2, as recited in Example 1).
[0053] Human 279 contains the following regions or other structural
features:
[0054] a GPCR domain (PFAM Accession Number PF00001) located at
about amino acid residues 34 to 286 of SEQ ID NO: 2, which includes
one predicted G-protein coupled receptors signature (PS00237) from
about amino acids 103 to 119;
[0055] 6 predicted Protein Kinase C phosphorylation sites (PS00005)
at about amino acids 89 to 91, 200 to 202, 214 to 216, 310 to 312,
328 to 330, and 334 to 336 of SEQ ID NO: 2;
[0056] 7 predicted transmembrane domains located at about amino
acids 23 to 43, 52 to 74, 92 to 113, 134 to 154, 186 to 205, 225 to
245, and 272 to 289 of SEQ ID NO: 2;
[0057] one predicted N-terminal, extracellular domain located at
about amino acids 1 to 22 of SEQ ID NO: 2;
[0058] one predicted C-terminal, cytoplasmic domain located at
about amino acids 290 to 362 of SEQ ID NO: 2;
[0059] three extracellular loops located at about amino acids 75 to
91, 155 to 185 and 246 to 271 of SEQ ID NO: 2;
[0060] three cytoplasmic loops located at about amino acids 44 to
51, 114 to 133 and 206 to 224 of SEQ ID NO: 2;
[0061] 3 predicted Casein Kinase II phosphorylation sites (PS00006)
located at about amino acids 13 to 16, 60 to 63, and 348 to 351 of
SEQ ID NO: 2;
[0062] 1 predicted cAMP/cGMP-dependent protein kinase
phosphorylation sites (PS00004) located at about amino acids 330 to
333 of SEQ IT) NO: 2;
[0063] 5 predicted N-glycosylation sites (PS00001) from about amino
acids 3 to 6, 58 to 61, 164 to 167, 318 to 321, and 332 to 335 of
SEQ ID NO: 2; and
[0064] 4 predicted N-myristylation sites (PS00008) from about amino
acids 31 to 36, 203 to 208, 210 to 215, and 340 to 345 of SEQ ID
NO: 2.
[0065] For general information regarding PFAM identifiers, PS
prefix and PF prefix domain identification numbers, refer to
Sonnhammer et al. (1997) Protein 28:405-420 and
http://www.psc.edu/general/software/package- s/pfam/pfam.html.
[0066] The 279 protein contains a significant number of structural
characteristics in common with members of the GPCR family. The term
"family" when referring to the protein and nucleic acid molecules
of the invention means two or more proteins or nucleic acid
molecules having a common structural domain or motif and having
sufficient amino acid or nucleotide sequence homology as defined
herein. Such family members can be naturally or non-naturally
occurring and can be from either the same or different species. For
example, a family can contain a first protein of human origin as
well as other distinct proteins of human origin, or alternatively,
can contain homologues of non-human origin, e.g., rat or mouse
proteins. Members of a family can also have common functional
characteristics.
[0067] The G-protein coupled receptor family of proteins is an
extensive group of proteins, which transduce extracellular signals
triggered by, e.g., hormones, neurotransmitters, odorants and
light, by interaction with guanine nucleotide-binding (G) proteins.
G-protein coupled receptors typically have seven hydrophobic
membrane spanning regions. The N-terminus of G-protein coupled
receptors is typically located on the extracellular side of the
membrane and is often glycosylated, while the C-terminus is
cytoplasmic and generally phosphorylated. Three extracellular loops
alternate with three intracellular loops to link the seven
transmembrane regions. Some G-protein coupled receptors possess a
signal peptide. Generally, the most conserved portions of G-protein
coupled receptors are the transmembrane regions and the first two
cytoplasmic loops. A conserved acidic-arginine-aromatic triplet is
present in the N-terminal extremity of the second cytoplasmic loop
and may be implicated in the interaction with G proteins. An
alignment of the transmembrane domains of 44 representative GPCRs
can be found at
<http://mgdkk1.nid11.nih.gov:8000/extended.html>.
[0068] The 279 polypeptide is similar in amino acid sequence to the
sequence of the G protein-coupled receptor, GPR4, disclosed in
Accession No. L36148; Heiber, M. et al. (1995) DNA Cell Biol.
14(1), 25-35. Human GPR4 was localized to chromosome 19
(q13.2-q13.3).
[0069] Accordingly, a 279 polypeptide can include a "GPCR domain"
or regions homologous with a "GPCR domain".
[0070] As used herein, the term "GPCR domain" includes an amino
acid sequence of about 100 to 350 amino acid residues in length and
having a bit score for the alignment of the sequence to the GPCR
domain profile (Pfam HMM) of at least 130. Preferably, a GPCR
domain includes at least about 200 to 300 amino acids, more
preferably about 225 to 275 amino acid residues, or about 250 to
255 amino acids and has a bit score for the alignment of the
sequence to the GPCR domain (HMM) of at least 150, 170, 200 or
greater. The GPCR domain (HMM) has been assigned the PFAM Accession
Number PF00001 (http//genome.wustl.edu/Pfam/.html) An alignment of
the GPCR domain (amino acids 34 to 286 of SEQ ID NO: 2) of human
279 with a consensus amino acid sequence (SEQ ID NO: 4) derived
from a hidden Markov model is depicted in FIG. 2.
[0071] In a preferred embodiment 279 polypeptide or protein has a
"GPCR domain" or a region which includes at least about 200 to 300
more preferably about 225 to 275 or 300 amino acid residues and has
at least about 70% 80% 90% 95%, 99%, or 100% homology with a "GPCR
domain," e.g., the GPCR domain of human 279 (e.g., residues 34 to
286 of SEQ ID NO: 2).
[0072] To identify the presence of a "GPCR" domain in a 279 protein
sequence, and make the determination that a polypeptide or protein
of interest has a particular profile, the amino acid sequence of
the protein can be searched against the Pfam database of HMMs
(e.g., the Pfam database, release 2.1) using the default parameter
(http://www.sanger.ac.uk/Software/Pfam/HMM_search). For example,
the hmmsf program, which is available as part of the HMMER package
of search programs, is a family specific default program for
MILPAT0063 and a score of 15 is the default threshold score for
determining a hit. Alternatively, the threshold score for
determining a hit can be lowered (e.g., to 8 bits). A description
of the Pfam database can be found in Sonhammer et al. (1997)
Proteins 28(3):405-420 and a detailed description of HMMs can be
found, for example, in Gribskov et al.(1990) Meth. Enzymol.
183:146-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci. USA
84:4355-4358; Krogh et al.(1994) J. Mol. Biol. 235:1501-1531; and
Stultz et al.(1993) Protein Sci. 2:305-314, the contents of which
are incorporated herein by reference. A search was performed
against the HMM database resulting in the identification of a
"GPCR" domain in the amino acid sequence of human 279 at about
residues 34 to 286 of SEQ ID NO: 2 (see FIG. 2).
[0073] A 279 family member can include at least one, two, three,
four, five, six, or preferably, seven transmembrane domains. As
used herein, the term "transmembrane domain" includes an amino acid
sequence of about 15 amino acid residues in length that spans the
plasma membrane. More preferably, a transmembrane domain includes
about at least 20, 23, 24, 25, 30 or 35 amino acid residues and
spans the plasma membrane. Transmembrane domains are rich in
hydrophobic residues, and typically have an .alpha.-helical
structure. In a preferred embodiment, at least 50%, 60%, 70%, 80%,
90%, 95% or more of the amino acids of a transmembrane domain are
hydrophobic, e.g., leucines, isoleucines, tyrosines, or
tryptophans. Transmembrane domains are described in, for example,
htto://pfam.wustl.edu/cgi-bin/getdesc?name=7tm-1, and Zagotta W. N.
et al, (1996) Annual Rev. Neurosci. 19: 235-63, the contents of
which are incorporated herein by reference. Amino acid residues
23-43, 52-74, 92-113, 134-154, 186-205, 225-245 and 272-289 of SEQ
ID NO: 2 comprise transmembrane domains in a 279 protein.
[0074] In another embodiment, a 65494 protein includes at least
one, two, or three extracellular loops. As defined herein, the term
"loop" includes an amino acid sequence having a length of at least
about 10, preferably about 12-35, more preferably about 15-32, and
even more preferably about 16-30 amino acid residues, and has an
amino acid sequence that connects two transmembrane domains within
a protein or polypeptide. Accordingly, the N-terminal amino acid of
a loop is adjacent to a C-terminal amino acid of a transmembrane
domain in a naturally-occurring 279 or 279-like molecule, and the
C-terminal amino acid of a loop is adjacent to an N-terminal amino
acid of a transmembrane domain in a naturally-occurring 279 or
279-like molecule. As used herein, an "extracellular loop" includes
an amino acid sequence located outside of a cell, or
extracellularly. For example, an extracellular loop can be found at
about amino acids 75-91, 155-185, and 245-271 of SEQ ID NO: 2.
[0075] In a preferred embodiment, a 279 polypeptide or protein has
at least one extracellular loop or a region which includes at least
about 10, preferably about 12-35, more preferably about 15-32, and
even more preferably about 16-30 amino acid residues and has at
least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with an
"extracellular loop," e.g., at least one extracellular loop of
human 279 (e.g., residues 75-91, 155-185, and 245-271 of SEQ ID NO:
2).
[0076] In another embodiment, a 279 protein includes at least one,
two or three cytoplasmic loops, also referred to herein as a
cytoplasmic domain. As used herein, a "cytoplasmic loop" includes
an amino acid sequence having a length of at least about 5,
preferably about 5-25, more preferably about 7-20, and most
preferably about 7-19 amino acid residues located within a cell or
within the cytoplasm of a cell. For example, a cytoplasmic loop is
found at about amino acids 44-51, 114-133, and 206-221 of SEQ ID
NO: 2.
[0077] In a preferred embodiment, a 279 polypeptide or protein has
at least one cytoplasmic loop or a region which includes at least
about 4, preferably about at least about 5, preferably about 5-25,
more preferably about 7-20, and most preferably about 7-19 amino
acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or
100% homology with an "cytoplasmic loop," e.g., at least one
cytoplasmic loop of human 65494 (e.g., residues 44-51, 114-133, and
206-221 of SEQ ID NO: 2).
[0078] When located at the N-terminal domain the extracellular
domain is referred to herein as an "N-terminal extracellular
domain", or as an "N-terminal extracellular loop" in the amino acid
sequence of the protein. As used herein, an "N-terminal
extracellular domain" includes an amino acid sequence having about
1-100, preferably about 1-50, more preferably about 1-30, more
preferably about 1-28, more preferably about 1-25, or even more
preferably about 1-22 amino acid residues in length and is located
outside of a cell or extracellularly. The C-terminal amino acid
residue of a "N-terminal extracellular domain" is adjacent to an
N-terminal amino acid residue of a transmembrane domain in a
naturally-occurring 279-like protein. For example, an N-terminal
cytoplasmic domain is located at about amino acid residues 1-22 of
SEQ ID NO: 2.
[0079] In a preferred embodiment, a 279 polypeptide or protein has
an "N-terminal extracellular domain" or a region which includes at
least about 1-100, preferably about 1-50, more preferably about
1-30, more preferably about 1-28, more preferably about 1-25, or
even more preferably about 1-22 amino acid residues and has at
least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with an
"N-terminal extracellular domain," e.g., the N-terminal
extracellular domain of human 279 (e.g., residues 1-22 of SEQ ID
NO: 2). Preferably, the N-terminal extracellular domain is capable
of interacting (e.g., binding to) with an extracellular signal, for
example, a ligand or a cell surface receptor.
[0080] In another embodiment, a 279 protein includes a "C-terminal
cytoplasmic domain", also referred to herein as a C-terminal
cytoplasmic tail, in the sequence of the protein. As used herein, a
"C-terminal cytoplasmic domain" includes an amino acid sequence
having a length of at least about 50, preferably about 50-100, more
preferably about 60-80, even more preferably, 72 amino acid
residues and is located within a cell or within the cytoplasm of a
cell. Accordingly, the N-terminal amino acid residue of a
"C-terminal cytoplasmic domain" is adjacent to a C-terminal amino
acid residue of a transmembrane domain in a naturally-occurring
279-like protein. For example, a C-terminal cytoplasmic domain is
found at about amino acid residues 290 to 362 of SEQ ID NO: 2.
[0081] In a preferred embodiment, a 279 polypeptide or protein has
a C-terminal cytoplasmic domain or a region which includes at least
about 50, preferably about 50-100, more preferably about 60-80,
even more preferably, 72 amino acid residues and has at least about
60%, 70% 80% 90% 95%, 99%, or 100% homology with an "C-terminal
cytoplasmic domain," e.g., the C-terminal cytoplasmic domain of
human 279 (e.g., residues 290 to 362 of SEQ ID NO: 2).
[0082] Furthermore, a 279 family member can include at least one,
two, three, four, five, preferably six protein kinase C
phosphorylation sites (PS00005); at least one, two, and preferably
three predicted casein kinase II phosphorylation sites (PS00006);
and at least one, two, three, and preferably four predicted
N-myristylation sites (PS00008).
[0083] As the 279 polypeptides of the invention may modulate
279-mediated activities, they may be useful as of for developing
novel diagnostic and therapeutic agents for 279-mediated or related
disorders, as described below.
[0084] As used herein, a "279 activity", "biological activity of
279" or "functional activity of 279" , refers to an activity
exerted by a 279 protein, polypeptide or nucleic acid molecule. For
example, a 279 activity can be an activity exerted by 279 in a
physiological milieu on, e.g., a 279-responsive cell or on a 279
substrate, e.g., a protein substrate. A 279 activity can be
determined in vivo or in vitro. In one embodiment, a 279 activity
is a direct activity, such as an association with a 279 target
molecule. A "target molecule" or "binding partner" is a molecule
with which a 279 protein binds or interacts in nature. A 279
activity can also be an indirect activity, e.g., a cellular
signaling activity mediated by interaction of the 279 protein with
a 279 receptor. The features of the 279 molecules of the present
invention can provide similar biological activities as GPCR family
members.
[0085] The response mediated by a 279 protein can depend upon the
type of cell. For example, in some cells, binding of a ligand,
e.g., a phospholipid (e.g., a sphingosylphosphorylcholine or a
lysophosphatidylcholine) to a 279 protein may stimulate an activity
such as release of compounds, gating of a channel, cellular
adhesion, migration, differentiation, etc., through
phosphatidylinositol, calcium signaling, or cyclic AMP metabolism
and turnover while in other cells, the binding of the ligand will
produce a different result. Regardless of the cellular
activity/response modulated by the receptor protein, it is
universal that the protein is a GPCR and interacts with G proteins
to produce one or more secondary signals, in a variety of
intracellular signal transduction pathways, e.g., through
phosphatidylinositol or cyclic AMP metabolism and turnover, in a
cell. As used herein, a "signaling transduction pathway" refers to
the modulation (e.g., stimulation or inhibition) of a cellular
function/activity upon the binding of a ligand to the GPCR (279
protein). Examples of such functions include mobilization of
intracellular molecules that participate in a signal transduction
pathway, e.g., phosphatidylinositol 4,5-bisphosphate (PIP.sub.2),
inositol 1,4,5-triphosphate (IP.sub.3) and adenylate cyclase.
[0086] As used herein, "phosphatidylinositol turnover and
metabolism" refers to the molecules involved in the turnover and
metabolism of phosphatidylinositol 4,5-bisphosphate (PIP.sub.2) as
well as to the activities of these molecules. PIP.sub.2 is a
phospholipid found in the cytosolic leaflet of the plasma membrane.
Binding of ligand to the receptor activates, in some cells, the
plasma-membrane enzyme phospholipase C that in turn can hydrolyze
PIP.sub.2 to produce 1,2-diacylglycerol (DAG) and inositol
1,4,5-triphosphate (IP.sub.3). Once formed IP.sub.3 can diffuse to
the endoplasmic reticulum surface where it can bind an IP.sub.3
receptor, e.g., a calcium channel protein containing an IP.sub.3
binding site. IP.sub.3 binding can induce opening of the channel,
allowing calcium ions to be released into the cytoplasm. IP.sub.3
can also be phosphorylated by a specific kinase to form inositol
1,3,4,5-tetraphosphate (IP.sub.4), a molecule which can cause
calcium entry into the cytoplasm from the extracellular medium.
IP.sub.3 and IP.sub.4can subsequently be hydrolyzed very rapidly to
the inactive products inositol 1,4-biphosphate (IP.sub.2) and
inositol 1,3,4-triphosphate, respectively. These inactive products
can be recycled by the cell and used to synthesize PIP.sub.2. The
other second messenger produced by the hydrolysis of PIP.sub.2,
namely 1,2-diacylglycerol (DAG), remains in the cell membrane where
it can serve to activate the enzyme protein kinase C. Protein
kinase C is usually found soluble in the cytoplasm of the cell, but
upon an increase in the intracellular calcium concentration, this
enzyme can move to the plasma membrane where it may be activated by
DAG. The activation of protein kinase C in different cells results
in various cellular responses such as the phosphorylation of
glycogen synthase, or the phosphorylation of various transcription
factors, e.g., NF-.kappa.B. The language "phosphatidylinositol
activity", as used herein, refers to an activity of PIP.sub.2 or
one of its metabolites.
[0087] Another signaling pathway in which the receptor may
participate is the cAMP turnover pathway. As used herein, "cyclic
AMP turnover and metabolism" refers to the molecules involved in
the turnover and metabolism of cyclic AMP (cAMP) as well as to the
activities of these molecules. Cyclic AMP is a second messenger
produced in response to ligand-induced stimulation of certain G
protein coupled receptors. In the cAMP signaling pathway, binding
of a ligand to a GPCR can lead to the activation of the enzyme
adenyl cyclase, which catalyzes the synthesis of cAMP. The newly
synthesized cAMP can in turn activate a cAMP-dependent protein
kinase. This activated kinase can phosphorylate a voltage-gated
potassium channel protein, or an associated protein, and lead to
the inability of the potassium channel to open during an action
potential. The inability of the potassium channel to open results
in a decrease in the outward flow of potassium, which normally
repolarizes the membrane of a neuron, leading to prolonged membrane
depolarization.
[0088] Given the significant structural similarity between the 279
polypeptide and GPR4, particularly in the ligand binding
extracellular domain, it is likely that these two receptors bind to
similar ligands and stimulate similar signal transduction pathways.
Phospholipids such as sphingolipids, e.g.,
sphingosylphosphorylcholine, and lysophosphatidylcholine are
ligands for the G protein couled receptor GPR4 (Zhu, K. et al.
(2001) JBC, in press). Binding of these phospholipids to
transfected cells overexpressing the GPR4-receptor leads to an
increase in the intracellular calcium level, phosphorylation of ERK
(cell proliferation pathway), DNA synthesis, and cell migration.
All of these effects are relevant to the pro-angiogenic function of
279 polyeptides described herein. For example, cell proliferation
and migration are directly linked to angiogenesis. Increased
intracellular calcium can lead to the activation of downstream
mediators of angiogenic signals, including eNOS (Bauer, K S et al
(1999) J. Pharmacology and Experimental Therapeutics 292: 31-37).
Carboxyamido-triazole inhibits angiogenesis by blocking the
calcium-mediated nitric-oxide synthase-vascular endothelial growth
factor pathway.
[0089] Phospholipids, e.g., sphingolipids (e.g.,
sphingosylphosphorylcholi- ne) and lysophospholipids, have been
shown to induce a number of cellular responses in endothelial
cells, including endothelial cell migration, morphogenesis, and
capillary-like tube formation (Boguslawski, G (2000) Biochem
Biophys Res Commun 272(2): 603-609); cell proliferation (Nofer, J-R
(2000) Biochemistry 39(49): 15199-15207); survival/anti-apoptotic
effect on endothelial cells, mediated through AKT, and similar to
established angiogenic factors such as VEGF and angiopoietin-1
(Nofer, J-R et al (2001) supra); nitric oxide (NO) production by
eNOS (Mogami, K (1999) FEBS Lett 457(3): 375); cytosolic Ca(2+)
elevation in endothelial cells and causes endothelium-dependent
relaxation through nitric oxide production in bovine coronary
artery (Lee, P C (1999) Am J Physiol 277 (4 Pt 2): H1600-H1608);
increased expression of IL-8, a pro-angiogenic cytokine, in ovarian
cancer cells (Schwartz, B M et al (2001) Gynecol Oncol 81(2):
291-300; Yoshida, S. et al (1997) Mol Cell Biol 17(7): 4015-4023);
induction of Cox-2 gene expression, which is pro-angiogenic, in
endothelial cells (Rikitake, Y (2001) Biochem Biophy Res Commun 281
(5): 1291-1297; Gately, S. (2000) Cancer Metastasis Rev 19(1-2):
19-27); constriction of renal and mesenteric microvessels in vitro
(Todoroki-Ikeda, N (2000) FEBS Lett 482(1-2): 85-90); induction of
Ca(2+)-sensitization of vascular smooth muscle contraction. A
number of biological processes have been linked to NO production,
e.g., wound healing and angiogenesis (Murohara, T (1998) J Clin
Invest 101(11): 2567-2578).
[0090] Phospholipids, e.g., lysophosphatidylcholine, have also been
linked to the modulation of vascular tone (Vuong, T D (2001) Kidney
Int 60(3): 1088-1096; Froese, D E (1999) Mol Cell Biochem 197
(1-2): 1-6), and atherosclerosis (Aoyama, T (1999) J Mol Cell
Cardio 31(12): 2010-2114; Sugiyama, S (1998) Arterioscler Thromb
Vasc Biol 18(4): 568-576).
[0091] Accordingly, based on the high degree of sequence homology
between the 279 polypeptides described herein and human GPR4
(particularly in the ligand-binding extracellular portion), the 279
polypeptide are believed to act as receptors for phospholipids such
as sphingosylphosphorylcholine and lysophospholipids, and mediate a
wide variety of cellular activities including one or more of: (1)
regulating, sensing and/or transmitting an extracellular signal
(e.g., a ligand, e.g., a phospholigand (e.g., a sphingolipid, e.g.,
sphingosylphosphorylcholine, or lysophospholipid)) into a cell, for
example, an endothelial cell; (2) mobilizing an intracellular
molecule that participates in a signal transduction pathway (e.g.,
a calcium ion, adenylate cyclase or phosphatidylinositol
4,5-bisphosphate (PIP2), inositol 1,4,5-triphosphate (IP3)); (3)
modulating cell (e.g., endothelial cell) proliferation, e.g.,
synthesis of DNA; (4) modulating migration, differentiation; and/or
survival of a cell, e.g., an endothelial cell; (5) modulating
vascular tone, e.g., vasoconstriction and/or vasodilation; (6)
modulating nitric oxide production; (7) modulating capillary tube
formation; (8) modulating blood vessel formation and/or
maintenance; or (9) modulating atherogenesis.
[0092] As the 279 mRNA is expressed in a number of cardiovascular
tissues (e.g., coronary smooth muscle cells, human umbilical vein
endothelial cells and human and mouse vessels), it may play an
important role in mediating cardiovascular disorders, e.g. blood
vessel disorders. Upregulation of 279 mRNA expression under
conditions related to angiogenesis (e.g., endothelial proliferation
and growth factor treatment), together with upregulation of 279
during angiogenesis in vivo (e.g., hemangioma) suggest that GPCR is
active during angiogenesis, and that an agonist may increase
angiogenesis. Additionally, upregulation of 279 expression in
atherosclerotic segments of mouse aorta suggests that an antagonist
of 279 may have therapeutic benefit in patients with
atherosclerosis.
[0093] Thus, the 279 molecules can act as novel diagnostic targets
and therapeutic agents for controlling 279-mediated disorders, such
as disorders involving aberrant angiogenesis, e.g., cardiovascular
and blood vessel disorders (e.g., atherosclerosis and disorders
involving vascular tone), and cancers.
[0094] Examples of cellular proliferative and/or differentiative
disorders include cancer, e.g., carcinoma, sarcoma, metastatic
disorders or hematopoietic neoplastic disorders, e.g., leukemias. A
metastatic tumor can arise from a multitude of primary tumor types,
including but not limited to those of prostate, colon, lung, breast
and liver origin.
[0095] As used herein, the terms "cancer", "hyperproliferative" and
"neoplastic" refer to cells having the capacity for autonomous
growth. Examples of such cells include cells having an abnormal
state or condition characterized by rapidly proliferating cell
growth. Hyperproliferative and neoplastic disease states may be
categorized as pathologic, i.e., characterizing or constituting a
disease state, or may be categorized as non-pathologic, i.e., a
deviation from normal but not associated with a disease state. The
term is meant to include all types of cancerous growths or
oncogenic processes, metastatic tissues or malignantly transformed
cells, tissues, or organs, irrespective of histopathologic type or
stage of invasiveness. "Pathologic hyperproliferative" cells occur
in disease states characterized by malignant tumor growth. Examples
of non-pathologic hyperproliferative cells include proliferation of
cells associated with wound repair.
[0096] The terms "cancer" or "neoplasms" include malignancies of
the various organ systems, such as affecting lung, breast, thyroid,
lymphoid, gastrointestinal, and genito-urinary tract, as well as
adenocarcinomas which include malignancies such as most colon
cancers, renal-cell carcinoma, prostate cancer and/or testicular
tumors, non-small cell carcinoma of the lung, cancer of the small
intestine and cancer of the esophagus.
[0097] The term "carcinoma" is art recognized and refers to
malignancies of epithelial or endocrine tissues including
respiratory system carcinomas, gastrointestinal system carcinomas,
genitourinary system carcinomas, testicular carcinomas, breast
carcinomas, prostatic carcinomas, endocrine system carcinomas, and
melanomas. Exemplary carcinomas include those forming from tissue
of the cervix, lung, prostate, breast, head and neck, colon and
ovary. The term also includes carcinosarcomas, e.g., which include
malignant tumors composed of carcinomatous and sarcomatous tissues.
An "adenocarcinoma" refers to a carcinoma derived from glandular
tissue or in which the tumor cells form recognizable glandular
structures.
[0098] The term "sarcoma" is art recognized and refers to malignant
tumors of mesenchymal derivation.
[0099] Examples of cellular proliferative and/or differentiative
disorders of the colon include, but are not limited to,
non-neoplastic polyps, adenomas, familial syndromes, colorectal
carcinogenesis, colorectal carcinoma, and carcinoid tumors.
[0100] Examples of cellular proliferative and/or differentiative
disorders of the liver include, but are not limited to, nodular
hyperplasias, adenomas, and malignant tumors, including primary
carcinoma of the liver and metastatic tumors.
[0101] Examples of cellular proliferative and/or differentiative
disorders of the breast include, but are not limited to,
proliferative breast disease including, e.g., epithelial
hyperplasia, sclerosing adenosis, and small duct papillomas;
tumors, e.g., stromal tumors such as fibroadenoma, phyllodes tumor,
and sarcomas, and epithelial tumors such as large duct papilloma;
carcinoma of the breast including in situ (noninvasive) carcinoma
that includes ductal carcinoma in situ (including Paget's disease)
and lobular carcinoma in situ, and invasive (infiltrating)
carcinoma including, but not limited to, invasive ductal carcinoma,
invasive lobular carcinoma, medullary carcinoma, colloid (mucinous)
carcinoma, tubular carcinoma, and invasive papillary carcinoma, and
miscellaneous malignant neoplasms. Disorders in the male breast
include, but are not limited to, gynecomastia and carcinoma.
[0102] Examples of cellular proliferative and/or differentiative
disorders of the lung include, but are not limited to, bronchogenic
carcinoma, including paraneoplastic syndromes, bronchioloalveolar
carcinoma, neuroendocrine tumors, such as bronchial carcinoid,
miscellaneous tumors, and metastatic tumors; pathologies of the
pleura, including inflammatory pleural effusions, noninflammatory
pleural effusions, pneumothorax, and pleural tumors, including
solitary fibrous tumors (pleural fibroma) and malignant
mesothelioma.
[0103] Further examples of cancers or neoplastic conditions, in
addition to the ones described above, include, but are not limited
to, a fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma,
osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,
lymphangiosarcoma, Iymphangioendotheliosarcoma, synovioma,
mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma,
gastric cancer, esophageal cancer, rectal cancer, pancreatic
cancer, ovarian cancer, prostate cancer, uterine cancer, cancer of
the head and neck, skin cancer, brain cancer, squamous cell
carcinoma, sebaceous gland carcinoma, papillary carcinoma,
papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma,
bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct
carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's
tumor, cervical cancer, testicular cancer, small cell lung
carcinoma, non-small cell lung carcinoma, bladder carcinoma,
epithelial carcinoma, glioma, astrocytoma, medulloblastoma,
craniopharyngioma, ependymoma, pinealoma, hemangioblastoma,
acoustic neuroma, oligodendroglioma, meningioma, melanoma,
neuroblastoma, retinoblastoma, leukemia, lymphoma, or Kaposi
sarcoma. Many such neoplastic conditions can progress to a
metastatic state, e.g., resulting in tumor cells moving to new
locations and forming metastatic tumors.
[0104] Additional examples of proliferative disorders include
hematopoietic neoplastic disorders. As used herein, the term
"hematopoietic neoplastic disorders" includes diseases involving
hyperplastic/neoplastic cells of hematopoietic origin. A
hematopoietic neoplastic disorder can arise from myeloid, lymphoid
or erythroid lineages, or precursor cells thereof. Preferably, the
diseases arise from poorly differentiated acute leukemias, e.g.,
erythroblastic leukemia and acute megakaryoblastic leukemia.
Additional exemplary myeloid disorders include, but are not limited
to, acute promyeloid leukemia (APML), acute myelogenous leukemia
(AML) and chronic myelogenous leukemia (CML) (reviewed in Vaickus,
L. (1991) Crit Rev. in Oncol./Hemotol. 11:267-97); lymphoid
malignancies include, but are not limited to acute lymphoblastic
leukemia (ALL) which includes B-lineage ALL and T-lineage ALL,
chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL),
hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM).
Additional forms of malignant lymphomas include, but are not
limited to non-Hodgkin lymphoma and variants thereof, peripheral T
cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous
T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF),
Hodgkin's disease and Reed-Sternberg disease.
[0105] As used herein, disorders involving the heart, or
"cardiovascular disease" or a "cardiovascular disorder" includes a
disease or disorder which affects the cardiovascular system, e.g.,
the heart, the blood vessels, and/or the blood. A cardiovascular
disorder can be caused by an imbalance in arterial pressure, a
malfunction of the heart, or an occlusion of a blood vessel, e.g.,
by a thrombus. A cardiovascular disorder includes, but is not
limited to disorders such as arteriosclerosis, atherosclerosis,
cardiac hypertrophy, ischemia reperfusion injury, restenosis,
arterial inflammation, vascular wall remodeling, ventricular
remodeling, rapid ventricular pacing, coronary microembolism,
tachycardia, bradycardia, pressure overload, aortic bending,
coronary artery ligation, vascular heart disease, valvular disease,
including but not limited to, valvular degeneration caused by
calcification, rheumatic heart disease, endocarditis, or
complications of artificial valves; atrial fibrillation, long-QT
syndrome, congestive heart failure, sinus node dysfunction, angina,
heart failure, hypertension, atrial fibrillation, atrial flutter,
pericardial disease, including but not limited to, pericardial
effusion and pericarditis; cardiomyopathies, e.g., dilated
cardiomyopathy or idiopathic cardiomyopathy, myocardial infarction,
coronary artery disease, coronary artery spasm, ischemic disease,
arrhythmia, sudden cardiac death, and cardiovascular developmental
disorders (e.g., arteriovenous malformations, arteriovenous
fistulae, raynaud's syndrome, neurogenic thoracic outlet syndrome,
causalgia/reflex sympathetic dystrophy, hemangioma, aneurysm,
cavernous angioma, aortic valve stenosis, atrial septal defects,
atrioventricular canal, coarctation of the aorta, ebsteins anomaly,
hypoplastic left heart syndrome, interruption of the aortic arch,
mitral valve prolapse, ductus arteriosus, patent foramen ovale,
partial anomalous pulmonary venous return, pulmonary atresia with
ventricular septal defect, pulmonary atresia without ventricular
septal defect, persistance of the fetal circulation, pulmonary
valve stenosis, single ventricle, total anomalous pulmonary venous
return, transposition of the great vessels, tricuspid atresia,
truncus arteriosus, ventricular septal defects). A cardiovasular
disease or disorder also can include an endothelial cell
disorder.
[0106] Disorders involving blood vessels include, but are not
limited to, responses of vascular cell walls to injury, such as
endothelial dysfunction and endothelial activation and intimal
thickening; vascular diseases including, but not limited to,
congenital anomalies, such as arteriovenous fistula,
atherosclerosis, and hypertensive vascular disease, such as
hypertension; inflammatory disease--the vasculitides, such as giant
cell (temporal) arteritis, Takayasu arteritis, polyarteritis nodosa
(classic), Kawasaki syndrome (mucocutaneous lymph node syndrome),
microscopic polyanglitis (microscopic polyarteritis,
hypersensitivity or leukocytoclastic anglitis), Wegener
granulomatosis, thromboanglitis obliterans (Buerger disease),
vasculitis associated with other disorders, and infectious
arteritis; Raynaud disease; aneurysms and dissection, such as
abdominal aortic aneurysms, syphilitic (luetic) aneurysms, and
aortic dissection (dissecting hematoma); disorders of veins and
lymphatics, such as varicose veins, thrombophlebitis and
phlebothrombosis, obstruction of superior vena cava (superior vena
cava syndrome), obstruction of inferior vena cava (inferior vena
cava syndrome), and lymphangitis and lymphedema; tumors, including
benign tumors and tumor-like conditions, such as hemangioma,
lymphangioma, glomus tumor (glomangioma), vascular ectasias, and
bacillary angiomatosis, and intermediate-grade (borderline
low-grade malignant) tumors, such as Kaposi sarcoma and
hemangloendothelioma, and malignant tumors, such as angiosarcoma
and hemangiopericytoma; and pathology of therapeutic interventions
in vascular disease, such as balloon angioplasty and related
techniques and vascular replacement, such as coronary artery bypass
graft surgery.
[0107] Upregulation of 279 mRNA expression in atherosclerotic
segments of mouse aorta suggests that an antagonist of 279 may have
therapeutic benefit in patients with atherosclerosis. As used
herein, the term "atherosclerosis" is intended to have its clinical
meaning. This term refers to a cardiovascular condition occurring
as a result of lesion formation in the arterial walls. The
narrowing is due to the formation of plaques or streaks in the
inner lining of the arteries. These plaques consist of foam cells
filled with modified low-density lipoproteins, oxidized-LDL,
decaying smooth muscle cells, fibrous tissue, clumps of blood
platelets, cholesterol, and sometimes calcium. They tend to form in
regions of disturbed blood flow and are found most often in people
with high concentrations of cholesterol in the bloodstream. The
number and thickness of plaques increase with age, causing loss of
the smooth lining of the blood vessels and encouraging the
formation of thrombi (blood clots). Sometimes fragments of thrombi
break off and form emboli, which travel through the bloodstream and
block smaller vessels. The thrombi or emboli can restrict the blood
supply to the heart, brain, kidney and other organs eventually
leading to end organ damage or death. The major causes of
atherosclerosis are hypercholesterolemia, hypoalphoproteinemia, and
hyperlipidemia marked by high circulating triglycerides in the
blood. These lipids are deposited in the arterial walls,
obstructing the blood flow and forming atherosclerotic plaques
leading to death.
[0108] As used herein the term "hypercholesterolemia" is a
condition with elevated levels of circulating total cholesterol,
LDL-cholesterol and VLDL-cholesterol as per the guidelines of the
Expert Panel Report of the National Cholesterol Educational Program
(NCEP) of Detection, Evaluation of Treatment of high cholesterol in
adults (see, Arch. Int. Med. (1988) 148, 36-39).
[0109] As used herein the term "hyperlipidemia" or "hyperlipemia"
is a condition where the blood lipid parameters are elevated in the
blood. This condition manifests an abnormally high concentration of
fats. The lipid fractions in the circulating blood are, total
cholesterol, low density lipoproteins, very low density
lipoproteins and triglycerides.
[0110] As used herein the term "lipoprotein" such as VLDL, LDL and
HDL, refers to a group of proteins found in the serum, plasma and
lymph and are important for lipid transport. The chemical
composition of each lipoprotein differs in that the HDL has a
higher proportion of protein versus lipid, whereas the VLDL has a
lower proportion of protein versus lipid.
[0111] As used herein, the term "triglyceride" means a lipid or
neutral fat consisting of glycerol combined with three fatty acid
molecules.
[0112] As used herein the term "xanthomatosis" is a disease
evidenced by a yellowish swelling or plaques in the skin resulting
from deposits of fat. The presence of xanthomas are usually
accompanied by raised blood cholesterol levels.
[0113] In those embodiments where treatment of a blood vessel
disorder, e.g., atherosclerosis, is desired, agents that modulate
279 expression or activity can be administered to a subject in
combination with a second agent, e.g., a cholesterol lowering
agent. Examples of cholesterol lowering agents include bile acid
sequestering resins (e.g. colestipol hydrochloride or
cholestyramine), fibric acid derivatives (e.g. clofibrate,
fenofibrate, or gemfibrozil), thiazolidenediones (e.g.,
troglitazone, pioglitazone, ciglitazone, englitazone,
rosiglitazone), or hydroxymethylglutaryl coenzyme A reductase
(HMG-CoA reductase) inhibitors (e.g. statins, such as fluvastatin
sodium, lovastatin, pravastatin sodium, simvastatin, atorvastatin
calcium, cerivastatin), an ApoAII-lowering agent, a VLDL lowering
agent, an ApoAI-stimulating agent, as well as inhibitors of,
nicotinic acid, niacin, or probucol. Preferred cholesterol lowering
agents include inhibitors of HMG-CoA reductase (e.g., statins),
nicotinic acid, and niacin. Preferably, the cholesterol lowering
agent results in a favorable plasma lipid profile (e.g., increased
HDL and/or reduced LDL).
[0114] In other embodiments, the agent(s) can be administered in
combination with an interventional procedure ("procedural vascular
trauma"). "Procedural vascular trauma" includes the effects of
surgical/medical-mechanical interventions into mammalian
vasculature, but does not include vascular trauma due to the
organic vascular pathologies listed hereinabove, or to unintended
traumas, such as due to an accident. Thus, procedural vascular
traumas within the scope of the present treatment method include
(1) organ grafting or transplantation, such as transplantation and
grafting of heart, kidney, liver and the like, e.g., involving
vessel anastomosis; (2) vascular surgery, such as coronary bypass
surgery, biopsy, heart valve replacement, atheroectomy,
thrombectomy, and the like; (3) transcatheter vascular therapies
(TVT) including angioplasty, e.g., laser angioplasty and PTCA
procedures discussed hereinbelow, employing balloon catheters, or
indwelling catheters; (4) vascular grafting using natural or
synthetic materials, such as in saphenous vein coronary bypass
grafts, dacron and venous grafts used for peripheral arterial
reconstruction, etc.; (5) placement of a mechanical shunt, such as
a PTFE hemodialysis shunt used for arteriovenous communications;
and (6) placement of an intravascular stent, which may be metallic,
plastic or a biodegradable polymer. See U.S. patent application
Ser. No. 08/389,712, filed Feb. 15, 1995, which is incorporated by
reference herein. For a general discussion of implantable devices
and biomaterials from which they can be formed, see H. Kambic et
al., "Biomaterials in Artificial Organs", Chem. Eng. News, 30 (Apr.
14, 1986), the disclosure of which is incorporated by reference
herein. Examples of interventional procedures, include but are not
limited to, angioplasty, placement of a shunt, stent, synthetic or
natural excision grafts, indwelling catheter, valve and other
implantable devices.
[0115] As used herein, an "angiogenic disorder" or an "endothelial
cell disorder" includes a disorder characterized by aberrant,
unregulated, or unwanted endothelial cell activity, e.g.,
proliferation, migration, angiogenesis, or vascularization; or
aberrant expression of cell surface adhesion molecules or genes
associated with angiogenesis, e.g., TIE-2, FLT and FLK. Endothelial
cell disorders include tumorigenesis, tumor metastasis, psoriasis,
diabetic retinopathy, endometriosis, Grave's disease, ischemic
disease (e.g., atherosclerosis), and chronic inflammatory diseases
(e.g., rheumatoid arthritis).
[0116] The 279 protein, fragments thereof, and derivatives and
other variants of the sequence in SEQ ID NO: 2 thereof are
collectively referred to as "polypeptides or proteins of the
invention" or "279 polypeptides or proteins". Nucleic acid
molecules encoding such polypeptides or proteins are collectively
referred to as "nucleic acids of the invention" or "279 nucleic
acids." 279 molecules refer to 279 nucleic acids, polypeptides, and
antibodies.
[0117] As used herein, the term "nucleic acid molecule" includes
DNA molecules (e.g., a cDNA or genomic DNA), RNA molecules (e.g.,
an mRNA) and analogs of the DNA or RNA. A DNA or RNA analog can be
synthesized from nucleotide analogs. The nucleic acid molecule can
be single-stranded or double-stranded, but preferably is
double-stranded DNA.
[0118] The term "isolated nucleic acid molecule" or "purified
nucleic acid molecule" includes nucleic acid molecules that are
separated from other nucleic acid molecules present in the natural
source of the nucleic acid. For example, with regards to genomic
DNA, the term "isolated" includes nucleic acid molecules which are
separated from the chromosome with which the genomic DNA is
naturally associated. Preferably, an "isolated" nucleic acid is
free of sequences which naturally flank the nucleic acid (i.e.,
sequences located at the 5' and/or 3' ends of the nucleic acid) in
the genomic DNA of the organism from which the nucleic acid is
derived. For example, in various embodiments, the isolated nucleic
acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1
kb, 0.5 kb or 0.1 kb of 5' and/or 3' nucleotide sequences which
naturally flank the nucleic acid molecule in genomic DNA of the
cell from which the nucleic acid is derived. Moreover, an
"isolated" nucleic acid molecule, such as a cDNA molecule, can be
substantially free of other cellular material, or culture medium
when produced by recombinant techniques, or substantially free of
chemical precursors or other chemicals when chemically
synthesized.
[0119] As used herein, the term "hybridizes under low stringency,
medium stringency, high stringency, or very high stringency
conditions" describes conditions for hybridization and washing.
Guidance for performing hybridization reactions can be found in
Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.
(1989), 6.3.1-6.3.6, which is incorporated by reference. Aqueous
and nonaqueous methods are described in that reference and either
can be used. Specific hybridization conditions referred to herein
are as follows: 1) low stringency hybridization conditions in
6.times.sodium chloride/sodium citrate (SSC) at about 45.degree.
C., followed by two washes in 0.2.times.SSC, 0.1% SDS at least at
50.degree. C. (the temperature of the washes can be increased to
55.degree. C. for low stringency conditions); 2) medium stringency
hybridization conditions in 6.times.SSC at about 45.degree. C.,
followed by one or more washes in 0.2.times.SSC, 0.1% SDS at
60.degree. C.; 3) high stringency hybridization conditions in
6.times.SSC at about 45.degree. C., followed by one or more washes
in 0.2.times.SSC, 0.1% SDS at 65.degree. C.; and preferably 4) very
high stringency hybridization conditions are 0.5M sodium phosphate,
7% SDS at 65.degree. C., followed by one or more washes at
0.2.times.SSC, 1% SDS at 65.degree. C. Very high stringency
conditions (4) are the preferred conditions and the ones that
should be used unless otherwise specified.
[0120] Preferably, an isolated nucleic acid molecule of the
invention that hybridizes under a stringency condition described
herein to the sequence of SEQ ID NO: 1 or SEQ ID NO: 3, corresponds
to a naturally-occurring nucleic acid molecule.
[0121] As used herein, a "naturally-occurring" nucleic acid
molecule refers to an RNA or DNA molecule having a nucleotide
sequence that occurs in nature. For example a naturally occurring
nucleic acid molecule can encode a natural protein.
[0122] As used herein, the terms "gene" and "recombinant gene"
refer to nucleic acid molecules which include at least an open
reading frame encoding a 279 protein. The gene can optionally
further include non-coding sequences, e.g., regulatory sequences
and introns. Preferably, a gene encodes a mammalian 279 protein or
derivative thereof.
[0123] An "isolated" or "purified" polypeptide or protein is
substantially free of cellular material or other contaminating
proteins from the cell or tissue source from which the protein is
derived, or substantially free from chemical precursors or other
chemicals when chemically synthesized. "Substantially free" means
that a preparation of 279 protein is at least 10% pure. In a
preferred embodiment, the preparation of 279 protein has less than
about 30%, 20%, 10% and more preferably 5% (by dry weight), of
non-279 protein (also referred to herein as a "contaminating
protein"), or of chemical precursors or non-279 chemicals. When the
279 protein or biologically active portion thereof is recombinantly
produced, it is also preferably substantially free of culture
medium, i.e., culture medium represents less than about 20%, more
preferably less than about 10%, and most preferably less than about
5% of the volume of the protein preparation. The invention includes
isolated or purified preparations of at least 0.01, 0.1, 1.0, and
10 milligrams in dry weight.
[0124] A "non-essential" amino acid residue is a residue that can
be altered from the wild-type sequence of 279 without abolishing or
substantially altering a 279 activity. Preferably the alteration
does not substantially alter the 279 activity, e.g., the activity
is at least 20%, 40%, 60%, 70% or 80% of wild-type. An "essential"
amino acid residue is a residue that, when altered from the
wild-type sequence of 279, results in abolishing a 279 activity
such that less than 20% of the wild-type activity is present. For
example, conserved amino acid residues in 279 are predicted to be
particularly unamenable to alteration.
[0125] A "conservative amino acid substitution" is one in which the
amino acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art. These families include
amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), nonpolar side
chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a
predicted nonessential amino acid residue in a 279 protein is
preferably replaced with another amino acid residue from the same
side chain family. Alternatively, in another embodiment, mutations
can be introduced randomly along all or part of a 279 coding
sequence, such as by saturation mutagenesis, and the resultant
mutants can be screened for 279 biological activity to identify
mutants that retain activity. Following mutagenesis of SEQ ID NO: 1
or SEQ ID NO: 3, the encoded protein can be expressed recombinantly
and the activity of the protein can be determined.
[0126] As used herein, a "biologically active portion" of a 279
protein includes a fragment of a 279 protein which participates in
an interaction, e.g., an intramolecular or an inter-molecular
interaction. An inter-molecular interaction can be a specific
binding interaction or an enzymatic interaction (e.g., the
interaction can be transient and a covalent bond is formed or
broken). An inter-molecular interaction can be between a 279
molecule and a non-279 molecule or between a first 279 molecule and
a second 279 molecule (e.g., a dimerization interaction).
Biologically active portions of a 279 protein include peptides
comprising amino acid sequences sufficiently homologous to or
derived from the amino acid sequence of the 279 protein, e.g., the
amino acid sequence shown in SEQ ID NO: 2, which include less amino
acids than the full length 279 proteins, and exhibit at least one
activity of a 279 protein. Typically, biologically active portions
comprise a domain or motif with at least one activity of the 279
protein, e.g., interacting (e.g., binding to) with an extracellular
ligand, e.g., a phospholipid. A biologically active portion of a
279 protein can be a polypeptide that is, for example, 10, 25, 50,
100, 200 or more amino acids in length. Biologically active
portions of a 279 protein can be used as targets for developing
agents which modulate a 279 mediated activity, e.g., interacting
(e.g., binding to) with an extracellular ligand, e.g., a
phospholipid.
[0127] Calculations of homology or sequence identity between
sequences (the terms are used interchangeably herein) are performed
as follows.
[0128] To determine the percent identity of two amino acid
sequences, or of two nucleic acid sequences, the sequences are
aligned for optimal comparison purposes (e.g., gaps can be
introduced in one or both of a first and a second amino acid or
nucleic acid sequence for optimal alignment and non-homologous
sequences can be disregarded for comparison purposes). In a
preferred embodiment, the length of a reference sequence aligned
for comparison purposes is at least 30%, preferably at least 40%,
more preferably at least 50%, 60%, and even more preferably at
least 70%, 80%, 90%, 100% of the length of the reference sequence.
The amino acid residues or nucleotides at corresponding amino acid
positions or nucleotide positions are then compared. When a
position in the first sequence is occupied by the same amino acid
residue or nucleotide as the corresponding position in the second
sequence, then the molecules are identical at that position (as
used herein amino acid or nucleic acid "identity" is equivalent to
amino acid or nucleic acid "homology").
[0129] The percent identity between the two sequences is a function
of the number of identical positions shared by the sequences,
taking into account the number of gaps, and the length of each gap,
which need to be introduced for optimal alignment of the two
sequences.
[0130] The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm. In a preferred embodiment, the percent
identity between two amino acid sequences is determined using the
Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm
which has been incorporated into the GAP program in the GCG
software package (available at http://www.gcg.com), using either a
Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14,
12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In
yet another preferred embodiment, the percent identity between two
nucleotide sequences is determined using the GAP program in the GCG
software package (available at http:H/www.gcg.com), using a
NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and
a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred
set of parameters (and the one that should be used unless otherwise
specified) are a Blossum 62 scoring matrix with a gap penalty of
12, a gap extend penalty of 4, and a frameshift gap penalty of
5.
[0131] The percent identity between two amino acid or nucleotide
sequences can be determined using the algorithm of E. Meyers and W.
Miller ((1989) CABIOS, 4:11-17) which has been incorporated into
the ALIGN program (version 2.0), using a PAM120 weight residue
table, a gap length penalty of 12 and a gap penalty of 4.
[0132] The nucleic acid and protein sequences described herein can
be used as a "query sequence" to perform a search against public
databases to, for example, identify other family members or related
sequences. Such searches can be performed using the NBLAST and
XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol.
Biol. 215:403-10. BLAST nucleotide searches can be performed with
the NBLAST program, score=100, wordlength=12 to obtain nucleotide
sequences homologous to 279 nucleic acid molecules of the
invention. BLAST protein searches can be performed with the XBLAST
program, score=50, wordlength=3 to obtain amino acid sequences
homologous to 279 protein molecules of the invention. To obtain
gapped alignments for comparison purposes, Gapped BLAST can be
utilized as described in Altschul et al., (1997) Nucleic Acids Res.
25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the
default parameters of the respective programs (e.g., XBLAST and
NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.
[0133] Particularly preferred 279 polypeptides of the present
invention have an amino acid sequence substantially identical to
the amino acid sequence of SEQ ID NO: 2. In the context of an amino
acid sequence, the term "substantially identical" is used herein to
refer to a first amino acid that contains a sufficient or minimum
number of amino acid residues that are i) identical to, or ii)
conservative substitutions of aligned amino acid residues in a
second amino acid sequence such that the first and second amino
acid sequences can have a common structural domain and/or common
functional activity. For example, amino acid sequences that contain
a common structural domain having at least about 60%, or 65%
identity, likely 75% identity, more likely 85%, 90%. 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 2 are termed
substantially identical.
[0134] In the context of nucleotide sequence, the term
"substantially identical" is used herein to refer to a first
nucleic acid sequence that contains a sufficient or minimum number
of nucleotides that are identical to aligned nucleotides in a
second nucleic acid sequence such that the first and second
nucleotide sequences encode a polypeptide having common functional
activity, or encode a common structural polypeptide domain or a
common functional polypeptide activity. For example, nucleotide
sequences having at least about 60%, or 65% identity, likely 75%
identity, more likely 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identity to SEQ ID NO: 1 or 3 are termed substantially
identical.
[0135] "Misexpression or aberrant expression", as used herein,
refers to a non-wildtype pattern of gene expression at the RNA or
protein level. It includes: expression at non-wild type levels,
i.e., over- or under-expression; a pattern of expression that
differs from wild type in terms of the time or stage at which the
gene is expressed, e.g., increased or decreased expression (as
compared with wild type) at a predetermined developmental period or
stage; a pattern of expression that differs from wild type in terms
of altered, e.g., increased or decreased, expression (as compared
with wild type) in a predetermined cell type or tissue type; a
pattern of expression that differs from wild type in terms of the
splicing size, translated amino acid sequence, post-transitional
modification, or biological activity of the expressed polypeptide;
a pattern of expression that differs from wild type in terms of the
effect of an environmental stimulus or extracellular stimulus on
expression of the gene, e.g., a pattern of increased or decreased
expression (as compared with wild type) in the presence of an
increase or decrease in the strength of the stimulus.
[0136] "Subject," as used herein, refers to human and non-human
animals. The term "non-human animals" of the invention includes all
vertebrates, e.g., mammals, such as non-human primates
(particularly higher primates), sheep, dog, rodent (e.g., mouse or
rat), guinea pig, goat, pig, cat, rabbits, cow, and non-mammals,
such as chickens, amphibians, reptiles, etc. In a preferred
embodiment, the subject is a human. In another embodiment, the
subject is an experimental animal or animal suitable as a disease
model.
[0137] A "purified preparation of cells", as used herein, refers to
an in vitro preparation of cells. In the case cells from
multicellular organisms (e.g., plants and animals), a purified
preparation of cells is a subset of cells obtained from the
organism, not the entire intact organism. In the case of
unicellular microorganisms (e.g., cultured cells and microbial
cells), it consists of a preparation of at least 10% and more
preferably 50% of the subject cells.
[0138] Various aspects of the invention are described in further
detail below.
[0139] Isolated Nucleic Acid Molecules
[0140] In one aspect, the invention provides, an isolated or
purified, nucleic acid molecule that encodes a 279 polypeptide
described herein, e.g., a full-length 279 protein or a fragment
thereof, e.g., a biologically active portion of 279 protein. Also
included is a nucleic acid fragment suitable for use as a
hybridization probe, which can be used, e.g., to identify a nucleic
acid molecule encoding a polypeptide of the invention, 279 mRNA,
and fragments suitable for use as primers, e.g., PCR primers for
the amplification or mutation of nucleic acid molecules.
[0141] In one embodiment, an isolated nucleic acid molecule of the
invention includes the nucleotide sequence shown in SEQ ID NO: 1,
or a portion of any of these nucleotide sequences. In one
embodiment, the nucleic acid molecule includes sequences encoding
the human 279 protein (i.e., "the coding region" of SEQ ID NO: 1,
as shown in SEQ ID NO: 3), as well as 5' untranslated sequences.
Alternatively, the nucleic acid molecule can include only the
coding region of SEQ ID NO: 1 (e.g., SEQ ID NO: 3) and, e.g., no
flanking sequences which normally accompany the subject sequence.
In another embodiment, the nucleic acid molecule encodes a sequence
corresponding to a fragment of the protein from about amino acid 34
to 286 of SEQ ID NO: 2.
[0142] In another embodiment, an isolated nucleic acid molecule of
the invention includes a nucleic acid molecule which is a
complement of the nucleotide sequence shown in SEQ ID NO: 1 or SEQ
ID NO: 3, or a portion of any of these nucleotide sequences. )In
other embodiments, the nucleic acid molecule of the invention is
sufficiently complementary to the nucleotide sequence shown in SEQ
ID NO: 1 or SEQ ID NO: 3, such that it can hybridize (e.g., under a
stringency condition described herein) to the nucleotide sequence
shown in SEQ ID NO: 1 or 3, thereby forming a stable duplex.
[0143] In one embodiment, an isolated nucleic acid molecule of the
present invention includes a nucleotide sequence which is at least
about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, homologous to the entire length of the
nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, or a
portion, preferably of the same length, of any of these nucleotide
sequences.
[0144] 279 Nucleic Acid Fragments
[0145] A nucleic acid molecule of the invention can include only a
portion of the nucleic acid sequence of SEQ ID NO: 1 or 3. For
example, such a nucleic acid molecule can include a fragment that
can be used as a probe or primer or a fragment encoding a portion
of a 279 protein, e.g., an immunogenic or biologically active
portion of a 279 protein. A fragment can comprise those nucleotides
of SEQ ID NO: 1, which encode a seven transmembrane domain of human
279, e.g., residues 34 to 286 of SEQ ID NO: 2 or a fragment
thereof. The nucleotide sequence determined from the cloning of the
279 gene allows for the generation of probes and primers designed
for use in identifying and/or cloning other 279 family members, or
fragments thereof, as well as 279 homologues, or fragments thereof,
from other species.
[0146] In another embodiment, a nucleic acid includes a nucleotide
sequence that includes part, or all, of the coding region and
extends into either (or both) the 5' or 3' noncoding region. Other
embodiments include a fragment that includes a nucleotide sequence
encoding an amino acid fragment described herein. Nucleic acid
fragments can encode a specific domain or site described herein or
fragments thereof, particularly fragments thereof which are at
least 50 amino acids in length. Fragments also include nucleic acid
sequences corresponding to specific amino acid sequences described
above or fragments thereof. Nucleic acid fragments should not to be
construed as encompassing those fragments that may have been
disclosed prior to the invention.
[0147] A nucleic acid fragment can include a sequence corresponding
to a domain, region, or functional site described herein. A nucleic
acid fragment can also include one or more domain, region, or
functional site described herein. Thus, for example, a 279 nucleic
acid fragment can include a sequence corresponding to a domain as
described herein, e.g., a seven transmembrane domain, a
transmembrane receptor domain, an extracellular loop, a cytoplasmic
loop, and/or a cytoplasmic domain.
[0148] 279 probes and primers are provided. Typically a
probe/primer is an isolated or purified oligonucleotide. The
oligonucleotide typically includes a region of nucleotide sequence
that hybridizes under a stringency condition described herein to at
least about 7, 12 or 15, preferably about 20 or 25, more preferably
about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides
of a sense or antisense sequence of SEQ ID NO: 1 or SEQ ID NO: 3,
or of a naturally occurring allelic variant or mutant of SEQ ID NO:
1 or SEQ ID NO: 3. Preferably, an oligonucleotide is less than
about 200, 150, 120, or 100 nucleotides in length.
[0149] In one embodiment, the probe or primer is attached to a
solid support, e.g., a solid support described herein.
[0150] One exemplary kit of primers includes a forward primer that
anneals to the coding strand and a reverse primer that anneals to
the non-coding strand. The forward primer can anneal to the start
codon, e.g., the nucleic acid sequence encoding amino acid residue
1 of SEQ ID NO: 2. The reverse primer can anneal to the ultimate
codon, e.g., the codon immediately before the stop codon, e.g., the
codon encoding amino acid residue 362 of SEQ ID NO: 2. In a
preferred embodiment, the annealing temperatures of the forward and
reverse primers differ by no more than 5, 4, 3, or 2.degree. C.
[0151] In a preferred embodiment the nucleic acid is a probe which
is at least 10, 12, 15, 18, 20 and less than 200, more preferably
less than 100, or less than 50, nucleotides in length. It should be
identical, or differ by 1, or 2, or less than 5 or 10 nucleotides,
from a sequence disclosed herein. If alignment is needed for this
comparison the sequences should be aligned for maximum homology.
"Looped" out sequences from deletions or insertions, or mismatches,
are considered differences.
[0152] A probe or primer can be derived from the sense or
anti-sense strand of a nucleic acid which encodes: one or more of
the seven transmembrane domains, from about amino acid residues
23-43, 52-74, 92-1113, 134-154, 186-205, 225-245 and 272-289 of SEQ
ID NO: 2; one or more of the extracellular loops, from about amino
acid residues 75-91, 155-185, and 245-271 of SEQ ID NO: 2; one or
more of the extracellular loops, from about amino acid residues
75-91, 155-185, and 245-271 of SEQ ID NO: 2; an N-terminal
extracellular domain from about amino acid residues 1 to 22 of SEQ
ID NO: 2; or a C-terminal cytoplasmic domain from about amino acids
290 to 362 of SEQ ID NO: 2.
[0153] In another embodiment a set of primers is provided, e.g.,
primers suitable for use in a PCR, which can be used to amplify a
selected region of a 279 sequence, e.g., a domain, region, site or
other sequence described herein. The primers should be at least 5,
10, or 50 base pairs in length and less than 100, or less than 200,
base pairs in length. The primers should be identical, or differs
by one base from a sequence disclosed herein or from a naturally
occurring variant. For example, primers suitable for amplifying all
or a portion of any of the following regions are provided: a 7
transmembrane domain from about amino acid 34 to 286 of SEQ ID NO:
2; one or more of the seven transmembrane domains, from about amino
acid residues 23-43, 52-74, 92-1113, 134-154, 186-205, 225-245 and
272-289 of SEQ ID NO: 2; one or more of the extracellular loops,
from about amino acid residues 75-91, 155-185, and 245-271 of SEQ
ID NO: 2; one or more of the extracellular loops, from about amino
acid residues 75-91, 155-185, and 245-271 of SEQ ID NO: 2; an
N-terminal extracellular domain from about amino acid residues 1 to
22 of SEQ ID NO: 2; or a C-terminal cytoplasmic domain from about
amino acids 290 to 362 of SEQ ID NO: 2.
[0154] A nucleic acid fragment can encode an epitope bearing region
of a polypeptide described herein.
[0155] A nucleic acid fragment encoding a "biologically active
portion of a 279 polypeptide" can be prepared by isolating a
portion of the nucleotide sequence of SEQ ID NO: 1 or 3, which
encodes a polypeptide having a 279 biological activity (e.g., the
biological activities of the 279 proteins are described herein),
expressing the encoded portion of the 279 protein (e.g., by
recombinant expression in vitro) and assessing the activity of the
encoded portion of the 279 protein. For example, a nucleic acid
fragment encoding a biologically active portion of 279 includes a 7
transmembrane domain, e.g., amino acid residues about 34 to 286 of
SEQ ID NO: 2. A nucleic acid fragment encoding a biologically
active portion of a 279 polypeptide, may comprise a nucleotide
sequence which is greater than 225 or more nucleotides in
length.
[0156] In preferred embodiments, a nucleic acid includes a
nucleotide sequence which is about 300, 400, 500, 600, 700, 800,
900, 1000, 1100, 1200, 1300 or more nucleotides in length and
hybridizes under a stringency condition described herein to a
nucleic acid molecule of SEQ ID NO: 1, or SEQ ID NO: 3.
[0157] In other embodiments, a nucleic acid fragment includes a
nucleotide sequence that encodes one or more of the last fifteen
amino acids of a 279-polypeptide, e.g., one or more of amino acids
347 to 362 of SEQ ID NO: 2.
[0158] 279 Nucleic Acid Variants
[0159] The invention further encompasses nucleic acid molecules
that differ from the nucleotide sequence shown in SEQ ID NO: 1 or
SEQ ID NO: 3. Such differences can be due to degeneracy of the
genetic code (and result in a nucleic acid which encodes the same
279 proteins as those encoded by the nucleotide sequence disclosed
herein. In another embodiment, an isolated nucleic acid molecule of
the invention has a nucleotide sequence encoding a protein having
an amino acid sequence which differs, by at least 1, but less than
5, 10, 20, 50, or 100 amino acid residues that shown in SEQ ID NO:
2. If alignment is needed for this comparison the sequences should
be aligned for maximum homology. The encoded protein can differ by
no more than 5, 4, 3, 2, or 1 amino acid. "Looped" out sequences
from deletions or insertions, or mismatches, are considered
differences.
[0160] Nucleic acids of the inventor can be chosen for having
codons, which are preferred, or non-preferred, for a particular
expression system. E.g., the nucleic acid can be one in which at
least one codon, at preferably at least 10%, or 20% of the codons
has been altered such that the sequence is optimized for expression
in E. coli, yeast, human, insect, or CHO cells.
[0161] Nucleic acid variants can be naturally occurring, such as
allelic variants (same locus), homologs (different locus), and
orthologs (different organism) or can be non naturally occurring.
Non-naturally occurring variants can be made by mutagenesis
techniques, including those applied to polynucleotides, cells, or
organisms. The variants can contain nucleotide substitutions,
deletions, inversions and insertions. Variation can occur in either
or both the coding and non-coding regions. The variations can
produce both conservative and non-conservative amino acid
substitutions (as compared in the encoded product).
[0162] In a preferred embodiment, the nucleic acid differs from
that of SEQ ID NO: 1 or 3, e.g., as follows: by at least one but
less than 10, 20, 30, or 40 nucleotides; at least one but less than
1%, 5%, 10% or 20% of the nucleotides in the subject nucleic acid.
The nucleic acid can differ by no more than 5, 4, 3, 2, or 1
nucleotide. If necessary for this analysis the sequences should be
aligned for maximum homology. "Looped" out sequences from deletions
or insertions, or mismatches, are considered differences.
[0163] Orthologs, homologs, and allelic variants can be identified
using methods known in the art. These variants comprise a
nucleotide sequence encoding a polypeptide that is 50%, at least
about 55%, typically at least about 70-75%, more typically at least
about 80-85%, and most typically at least about 90-95% or more
identical to the nucleotide sequence shown in SEQ ID NO: 2 or a
fragment of this sequence. Such nucleic acid molecules can readily
be identified as being able to hybridize under a stringency
condition described herein, to the nucleotide sequence shown in SEQ
ID NO 2 or a fragment of the sequence. Nucleic acid molecules
corresponding to orthologs, homologs, and allelic variants of the
279 cDNAs of the invention can further be isolated by mapping to
the same chromosome or locus as the 279 gene.
[0164] Preferred variants include those that are correlated with
regulating, sensing and/or transmitting an extracellular signal
into a cell; interacting with an extracellular signal or a cell
surface receptor; mobilizing an intracellular molecule that
participates in a signal transduction pathway; controlling
production or secretion of molecules; altering the structure of a
cellular component; modulating cell proliferation, e.g., synthesis
of DNA; and modulating cell migration, cell differentiation; and
cell survival.
[0165] Allelic variants of 279, e.g., human 279, include both
functional and non-functional proteins. Functional allelic variants
are naturally occurring amino acid sequence variants of the 279
protein within a population that maintain the ability to regulate,
sense and/or transmit an extracellular signal into a cell;
interacte with an extracellular signal or a cell surface receptor;
mobilize an intracellular molecule that participates in a signal
transduction pathway; control production or secretion of molecules;
alter the structure of a cellular component; modulate cell
proliferation, e:g., synthesis of DNA; and modulate cell migration,
cell differentiation; and cell survival.
[0166] Functional allelic variants will typically contain only
conservative substitution of one or more amino acids of SEQ ID NO:
2, or substitution, deletion or insertion of non-critical residues
in non-critical regions of the protein. Non-functional allelic
variants are naturally-occurring amino acid sequence variants of
the 279, e.g.,. human 279, protein within a population that do not
have the ability to regulate, sense and/or transmit an
extracellular signal into a cell; interacte with an extracellular
signal or a cell surface receptor; mobilize an intracellular
molecule that participates in a signal transduction pathway;
control production or secretion of molecules; alter the structure
of a cellular component; modulate cell proliferation, e.g.,
synthesis of DNA; and modulate cell migration, cell
differentiation; and cell survival. Non-functional allelic variants
will typically contain a non-conservative substitution, a deletion,
or insertion, or premature truncation of the amino acid sequence of
SEQ ID NO: 2, or a, substitution, insertion, or deletion in
critical residues or critical regions of the protein.
[0167] Moreover, nucleic acid molecules encoding other 279 family
members and, thus, which have a nucleotide sequence which differs
from the 279 sequences of SEQ ID NO: 1 or SEQ ID NO: 3 are intended
to be within the scope of the invention.
[0168] Antisense Nucleic Acid Molecules, Ribozymes and Modified 279
Nucleic Acid Molecules
[0169] In another aspect, the invention features, an isolated
nucleic acid molecule that is antisense to 279. An "antisense"
nucleic acid can include a nucleotide sequence which is
complementary to a "sense" nucleic acid encoding a protein, e.g.,
complementary to the coding strand of a double-stranded cDNA
molecule or complementary to an mRNA sequence. The antisense
nucleic acid can be complementary to an entire 279 coding strand,
or to only a portion thereof (e.g., the coding region of human 279
corresponding to SEQ ID NO: 3). In another embodiment, the
antisense nucleic acid molecule is antisense to a "noncoding
region" of the coding strand of a nucleotide sequence encoding 279
(e.g., the 5' and 3' untranslated regions).
[0170] An antisense nucleic acid can be designed such that it is
complementary to the entire coding region of 279 mRNA, but more
preferably is an oligonucleotide which is antisense to only a
portion of the coding or noncoding region of 279 mRNA. For example,
the antisense oligonucleotide can be complementary to the region
surrounding the translation start site of 279 mRNA, e.g., between
the -10 and +10 regions of the target gene nucleotide sequence of
interest. An antisense oligonucleotide can be, for example, about
7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or
more nucleotides in length.
[0171] An antisense nucleic acid of the invention can be
constructed using chemical synthesis and enzymatic ligation
reactions using procedures known in the art. For example, an
antisense nucleic acid (e.g., an antisense oligonucleotide) can be
chemically synthesized using naturally occurring nucleotides or
variously modified nucleotides designed to increase the biological
stability of the molecules or to increase the physical stability of
the duplex formed between the antisense and sense nucleic acids,
e.g., phosphorothioate derivatives and acridine substituted
nucleotides can be used. The antisense nucleic acid also can be
produced biologically using an expression vector into which a
nucleic acid has been subcloned in an antisense orientation (i.e.,
RNA transcribed from the inserted nucleic acid will be of an
antisense orientation to a target nucleic acid of interest,
described further in the following subsection).
[0172] The antisense nucleic acid molecules of the invention are
typically administered to a subject (e.g., by direct injection at a
tissue site), or generated in situ such that they hybridize with or
bind to cellular mRNA and/or genomic DNA encoding a 279 protein to
thereby inhibit expression of the protein, e.g., by inhibiting
transcription and/or translation. Alternatively, antisense nucleic
acid molecules can be modified to target selected cells and then
administered systemically. For systemic administration, antisense
molecules can be modified such that they specifically bind to
receptors or antigens expressed on a selected cell surface, e.g.,
by linking the antisense nucleic acid molecules to peptides or
antibodies which bind to cell surface receptors or antigens. The
antisense nucleic acid molecules can also be delivered to cells
using the vectors described herein. To achieve sufficient
intracellular concentrations of the anti sense molecules, vector
constructs in which the antisense nucleic acid molecule is placed
under the control of a strong pol II or pol III promoter are
preferred.
[0173] In yet another embodiment, the antisense nucleic acid
molecule of the invention is an .alpha.-anomeric nucleic acid
molecule. An .alpha.-anomeric nucleic acid molecule forms specific
double-stranded hybrids with complementary RNA in which, contrary
to the usual .beta.-units, the strands run parallel to each other
(Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The
antisense nucleic acid molecule can also comprise a
2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.
15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987)
FEBS Lett. 215:327-330).
[0174] In still another embodiment, an antisense nucleic acid of
the invention is a ribozyme. A ribozyme having specificity for a
279-encoding nucleic acid can include one or more sequences
complementary to the nucleotide sequence of a 279 cDNA disclosed
herein (i.e., SEQ ID NO: 1 or SEQ ID NO: 3), and a sequence having
known catalytic sequence responsible for mRNA cleavage (see U.S.
Pat. No. 5,093,246 or Haselhoff and Gerlach (1988) Nature
334:585-591). For example, a derivative of a Tetrahymena L-19 IVS
RNA can be constructed in which the nucleotide sequence of the
active site is complementary to the nucleotide sequence to be
cleaved in a 279-encoding mRNA. See, e.g., Cech et al. U.S. Pat.
No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742.
Alternatively, 279 mRNA can be used to select a catalytic RNA
having a specific ribonuclease activity from a pool of RNA
molecules. See, e.g., Bartel, D. and Szostak, J. W. (1993) Science
261:1411-1418.
[0175] 279 gene expression can be inhibited by targeting nucleotide
sequences complementary to the regulatory region of the 279 (e.g.,
the 279 promoter and/or enhancers) to form triple helical
structures that prevent transcription of the 279 gene in target
cells. See generally, Helene, C. (1991) AnticancerDrug Des.
6:569-84; Helene, C. i (1992) Ann. N.Y. Acad. Sci. 660:27-36; and
Maher, L. J. (1992) Bioassays 14:807-15. The potential sequences
that can be targeted for triple helix formation can be increased by
creating a so-called "switchback" nucleic acid molecule. Switchback
molecules are synthesized in an alternating 5'-3', 3'-5' manner,
such that they base pair with first one strand of a duplex and then
the other, eliminating the necessity for a sizeable stretch of
either purines or pyrimidines to be present on one strand of a
duplex.
[0176] The invention also provides detectably labeled
oligonucleotide primer and probe molecules. Typically, such labels
are chemiluminescent, fluorescent, radioactive, or
colorimetric.
[0177] A 279 nucleic acid molecule can be modified at the base
moiety, sugar moiety or phosphate backbone to improve, e.g., the
stability, hybridization, or solubility of the molecule. For
non-limiting examples of synthetic oligonucleotides with
modifications see Toulm (2001) Nature Biotech. 19:17 and Faria et
al. (2001) Nature Biotech. 19:40-44. Such phosphoramidite
oligonucleotides can be effective antisense agents.
[0178] For example, the deoxyribose phosphate backbone of the
nucleic acid molecules can be modified to generate peptide nucleic
acids (see Hyrup B. et al. (1996) Bioorganic & Medicinal
Chemistry 4: 5-23). As used herein, the terms "peptide nucleic
acid" or "PNA" refers to a nucleic acid mimic, e.g., a DNA mimic,
in which the deoxyribose phosphate backbone is replaced by a
pseudopeptide backbone and only the four natural nucleobases are
retained. The neutral backbone of a PNA can allow for specific
hybridization to DNA and RNA under conditions of low ionic
strength. The synthesis of PNA oligomers can be performed using
standard solid phase peptide synthesis protocols as described in
Hyrup B. et al. (1996) supra and Perry-O'Keefe et al. Proc. Natl.
Acad. Sci. 93: 14670-675.
[0179] PNAs of 279 nucleic acid molecules can be used in
therapeutic and diagnostic applications. For example, PNAs can be
used as antisense or antigene agents for sequence-specific
modulation of gene expression by, for example, inducing
transcription or translation arrest or inhibiting replication. PNAs
of 279 nucleic acid molecules can also be used in the analysis of
single base pair mutations in a gene, (e.g., by PNA-directed PCR
clamping); as `artificial restriction enzymes` when used in
combination with other enzymes, (e.g., S1 nucleases (Hyrup B. et
al. (1996) supra)); or as probes or primers for DNA sequencing or
hybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefe
supra).
[0180] In other embodiments, the oligonucleotide may include other
appended groups such as peptides (e.g., for targeting host cell
receptors in vivo), or agents facilitating transport across the
cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad.
Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad.
Sci. USA 84:648-652; PCT Publication No. W088/09810) or the
blood-brain barrier (see, e.g., PCT Publication No. W089/10134). In
addition, oligonucleotides can be modified with
hybridization-triggered cleavage agents (see, e.g., Krol et al.
(1988) Bio-Techniques 6:958-976) or intercalating agents. (see,
e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, the
oligonucleotide may be conjugated to another molecule, (e.g., a
peptide, hybridization triggered cross-linking agent, transport
agent, or hybridization-triggered cleavage agent).
[0181] The invention also includes molecular beacon oligonucleotide
primer and probe molecules having at least one region which is
complementary to a 279 nucleic acid of the invention, two
complementary regions one having a fluorophore and one a quencher
such that the molecular beacon is useful for quantitating the
presence of the 279 nucleic acid of the invention in a sample.
Molecular beacon nucleic acids are described, for example, in
Lizardi et al., U.S. Pat. No. 5,854,033; Nazarenko et al., U.S.
Pat. No. 5,866,336, and Livak et al., U.S. Pat. No. 5,876,930.
[0182] Isolated 279 Polypeptides
[0183] In another aspect, the invention features, an isolated 279
protein, or fragment, e.g., a biologically active portion, for use
as immunogens or antigens to raise or test (or more generally to
bind) anti-279 antibodies. 279 protein can be isolated from cells
or tissue sources using standard protein purification techniques.
279 protein or fragments thereof can be produced by recombinant DNA
techniques or synthesized chemically.
[0184] Polypeptides of the invention include those which arise as a
result of the existence of multiple genes, alternative
transcription events, alternative RNA splicing events, and
alternative translational and post-translational events. The
polypeptide can be expressed in systems, e.g., cultured cells,
which result in substantially the same post-translational
modifications present when expressed the polypeptide is expressed
in a native cell, or in systems which result in the alteration or
omission of post-translational modifications, e.g., glycosylation
or cleavage, present when expressed in a native cell.
[0185] In a preferred embodiment, a 279 polypeptide has one or more
of the following characteristics:
[0186] it has the ability to regulate, sense and/or transmit an
extracellular signal into a cell;
[0187] it has the ability to interact with (e.g., bind to) an
extracellular signal or a cell surface receptor;
[0188] it has the ability to mobilize an intracellular molecule
that participates in a signal transduction pathway;
[0189] it has the ability to modulate proliferation, migration,
differentiation and/or survival of a cell, e.g., an endothelial
cell;
[0190] it has the ability to modulate capillary tube formation;
[0191] it has the ability to modulate atherogenesis;
[0192] it can modulate NO production;
[0193] it can modulate calcium signaling;
[0194] it has a molecular weight, e.g., a deduced molecular weight,
preferably ignoring any contribution of post translational
modifications, amino acid composition or other physical
characteristic of a 279 polypeptide, e.g., a polyeptide of SEQ ID
NO: 2;
[0195] it has an overall sequence similarity of at least 60%, more
preferably-at least 70, 80, 90, or 95%, with a polypeptide of SEQ
ID NO: 2;
[0196] it can be found in endothelial cells and coronary smooth
muscle cells;
[0197] it has a GPCR domain which is preferably about 70%, 80%, 90%
or 95% with amino acid residues about 34 to 286 of SEQ ID NO: 2;
or
[0198] it can colocalize with a G protein;
[0199] In a preferred embodiment the 279 protein, or fragment
thereof, differs from the corresponding sequence in SEQ ID:2. In
one embodiment it differs by at least one but by less than 15, 10
or 5 amino acid residues. In another it differs from the
corresponding sequence in SEQ ID NO: 2 by at least one residue but
less than 20%, 15%, 10% or 5% of the residues in it differ from the
corresponding sequence in SEQ ID NO: 2. (If this comparison
requires alignment the sequences should be aligned for maximum
homology. "Looped" out sequences from deletions or insertions, or
mismatches, are considered differences.) The differences are,
preferably, differences or changes at a non essential residue or a
conservative substitution. In a preferred embodiment the
differences are not in the GPCR domain, e.g., amino acid residues
34 to 286. In another preferred embodiment one or more differences
are in the seven transmembrane domain e.g., amino acid residues 34
to 286 of SEQ ID NO: 2.
[0200] Other embodiments include a protein that contain one or more
changes in amino acid sequence, e.g., a change in an amino acid
residue which is not essential for activity. Such 279 proteins
differ in amino acid sequence from SEQ ID NO: 2, yet retain
biological activity.
[0201] In one embodiment, the protein includes an amino acid
sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%
or more homologous to SEQ ID NO: 2.
[0202] A 279 protein or fragment is provided which varies from the
sequence of SEQ ID NO: 2 in regions defined by amino acids about 1
to 33 and 287 to 362 by at least one but by less than 15, 10 or 5
amino acid residues in the protein or fragment but which does not
differ from SEQ ID NO: 2 in regions defined by amino acids about 34
to 286 of SEQ ID NO: 2. (If this comparison requires alignment the
sequences should be aligned for maximum homology. "Looped" out
sequences from deletions or insertions, or mismatches, are
considered differences.) In some embodiments the difference is at a
non-essential residue or is a conservative substitution, while in
others the difference is at an essential residue or is a
non-conservative substitution.
[0203] In one embodiment, a biologically active portion of a 279
protein includes a GPCR domain. Moreover, other biologically active
portions, in which other regions of the protein are deleted, can be
prepared by recombinant techniques and evaluated for one or more of
the functional activities of a native 279 protein.
[0204] In a preferred embodiment, the 279 protein has an amino acid
sequence shown in SEQ ID NO: 2. In other embodiments, the 279
protein is substantially identical to SEQ ID NO: 2. In yet another
embodiment, the 279 protein is substantially identical to SEQ ID
NO: 2 and retains the functional activity of the protein of SEQ ID
NO: 2, as described in detail in the subsections above.
[0205] In other embodiments, the 279 protein or fragment thereof
includes one or more residues from the last fifteen residues of SEQ
ID NO: 2, e.g., one or more residues from about amino acids 347 to
362 of SEQ ID NO: 2.
[0206] 279 Chimeric or Fusion Proteins
[0207] In another aspect, the invention provides 279 chimeric or
fusion proteins. As used herein, a 279 "chimeric protein" or
"fusion protein" includes a 279 polypeptide linked to a non-279
polypeptide. A "non-279 polypeptide" refers to a polypeptide having
an amino acid sequence corresponding to a protein which is not
substantially homologous to the 279 protein, e.g., a protein which
is different from the 279 protein and which is derived from the
same or a different organism. The 279 polypeptide of the fusion
protein can correspond to all or a portion e.g., a fragment
described herein of a 279 amino acid sequence. In a preferred
embodiment, a 279 fusion protein includes at least one (or two)
biologically active portion of a 279 protein. The non-279
polypeptide can be fused to the N-terminus or C-terminus of the 279
polypeptide.
[0208] The fusion protein can include a moiety that has a high
affinity for a ligand. For example, the fusion protein can be a
GST-279 fusion protein in which the 279 sequences are fused to the
C-terminus of the GST sequences. Such fusion proteins can
facilitate the purification of recombinant 279. Alternatively, the
fusion protein can be a 279 protein containing a heterologous
signal sequence at its N-terminus. In certain host cells (e.g.,
mammalian host cells), expression and/or secretion of 279 can be
increased through use of a heterologous signal sequence.
[0209] Fusion proteins can include all or a part of a serum
protein, e.g., an IgG constant region, or human serum albumin.
[0210] The 279 fusion proteins of the invention can be incorporated
into pharmaceutical compositions and administered to a subject in
vivo. The 279 fusion proteins can be used to affect the
bioavailability of a 279 substrate. 279 fusion proteins may be
useful therapeutically for the treatment of disorders caused by,
for example, (i) aberrant modification or mutation of a gene
encoding a 279 protein; (ii) mis-regulation of the 279 gene; and
(iii) aberrant post-translational modification of a 279
protein.
[0211] Moreover, the 279-fusion proteins of the invention can be
used as immunogens to produce anti-279 antibodies in a subject, to
purify 279 ligands and in screening assays to identify molecules
which inhibit the interaction of 279 with a 279 substrate.
[0212] Expression vectors are commercially available that already
encode a fusion moiety (e.g., a GST polypeptide). A 279-encoding
nucleic acid can be cloned into such an expression vector such that
the fusion moiety is linked in-frame to the 279 protein.
[0213] Variants of 279 Proteins
[0214] In another aspect, the invention also features a variant of
a 279 polypeptide, e.g., which functions as an agonist (mimetics)
or as an antagonist. Variants of the 279 proteins can be generated
by mutagenesis, e.g., discrete point mutation, the insertion or
deletion of sequences or the truncation of a 279 protein. An
agonist of the 279 proteins can retain substantially the same, or a
subset, of the biological activities of the naturally occurring
form of a 279 protein. An antagonist of a 279 protein can inhibit
one or more of the activities of the naturally occurring form of
the 279 protein by, for example, competitively modulating a
279-mediated activity of a 279 protein. Thus, specific biological
effects can be elicited by treatment with a variant of limited
function. Preferably, treatment of a subject with a variant having
a subset of the biological activities of the naturally occurring
form of the protein has fewer side effects in a subject relative to
treatment with the naturally occurring form of the 279 protein.
[0215] Variants of a 279 protein can be identified by screening
combinatorial libraries of mutants, e.g., truncation mutants, of a
279 protein for agonist or antagonist activity.
[0216] Libraries of fragments e.g., N terminal, C terminal, or
internal fragments, of a 279 protein coding sequence can be used to
generate a variegated population of fragments for screening and
subsequent selection of variants of a 279 protein. Variants in
which a cysteine residues is added or deleted or in which a residue
which is glycosylated is added or deleted are particularly
preferred.
[0217] Methods for screening gene products of combinatorial
libraries made by point mutations or truncation, and for screening
cDNA libraries for gene products having a selected property are
known in the art. Such methods are adaptable for rapid screening of
the gene libraries generated by combinatorial mutagenesis of 279
proteins. Recursive ensemble mutagenesis (REM), a new technique
which enhances the frequency of functional mutants in the
libraries, can be used in combination with the screening assays to
identify 279 variants (Arkin and Yourvan (1992) Proc. Natl. Acad.
Sci. USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering
6:327-331).
[0218] Cell based assays can be exploited to analyze a variegated
279 library. For example, a library of expression vectors can be
transfected into a cell line, e.g., a cell line, which ordinarily
responds to 279 in a substrate-dependent manner. The transfected
cells are then contacted with 279 and the effect of the expression
of the mutant on signaling by the 279 substrate can be detected,
e.g., by measuring cellular signaling activity, cellular survival,
or cellular migration or proliferation. Plasmid DNA can then be
recovered from the cells which score for inhibition, or
alternatively, potentiation of signaling by the 279 substrate, and
the individual clones further characterized.
[0219] In another aspect, the invention features a method of making
a 279 polypeptide, e.g., a peptide having a non-wild type activity,
e.g., an antagonist, agonist, or super agonist of a naturally
occurring 279 polypeptide, e.g., a naturally occurring 279
polypeptide. The method includes: altering the sequence of a 279
polypeptide, e.g., altering the sequence , e.g., by substitution or
deletion of one or more residues of a non-conserved region, a
domain or residue disclosed herein, and testing the altered
polypeptide for the desired activity.
[0220] In another aspect, the invention features a method of making
a fragment or analog of a 279 polypeptide a biological activity of
a naturally occurring 279 polypeptide. The method includes:
altering the sequence, e.g., by substitution or deletion of one or
more residues, of a 279 polypeptide, e.g., altering the sequence of
a non-conserved region, or a domain or residue described herein,
and testing the altered polypeptide for the desired activity.
[0221] Anti-279 Antibodies
[0222] In another aspect, the invention provides an anti-279
antibody, or a fragment thereof (e.g., an antigen-binding fragment
thereof). The term "antibody" as used herein refers to an
immunoglobulin molecule or immunologically active portion thereof,
i.e., an antigen-binding portion. As used herein, the term
"antibody" refers to a protein comprising at least one, and
preferably two, heavy (H) chain variable regions (abbreviated
herein as VH), and at least one and preferably two light (L) chain
variable regions (abbreviated herein as VL). The VH and VL regions
can be further subdivided into regions of hypervariability, termed
"complementarity determining regions" ("CDR"), interspersed with
regions that are more conserved, termed "framework regions" (FR).
The extent of the framework region and CDR's has been precisely
defined (see, Kabat, E. A., et al. (1991) Sequences of Proteins of
Immunological Interest, Fifth Edition, U.S. Department of Health
and Human Services, NIH Publication No. 91-3242, and Chothia, C. et
al. (1987) J. Mol. Biol. 196:901-917, which are incorporated herein
by reference). Each VH and VL is composed of three CDR's and four
FRs, arranged from amino-terminus to carboxy-terminus in the
following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
[0223] The anti-279 antibody can further include a heavy and light
chain constant region, to thereby form a heavy and light
immunoglobulin chain, respectively. In one embodiment, the antibody
is a tetramer of two heavy immunoglobulin chains and two light
immunoglobulin chains, wherein the heavy and light immunoglobulin
chains are inter-connected by, e.g., disulfide bonds. The heavy
chain constant region is comprised of three domains, CH1, CH2 and
CH3. The light chain constant region is comprised of one domain,
CL. The variable region of the heavy and light chains contains a
binding domain that interacts with an antigen. The constant regions
of the antibodies typically mediate the binding of the antibody to
host tissues or factors, including various cells of the immune
system (e.g., effector cells) and the first component (Clq) of the
classical complement system.
[0224] As used herein, the term "immunoglobulin" refers to a
protein consisting of one or more polypeptides substantially
encoded by immunoglobulin genes. The recognized human
immunoglobulin genes include the kappa, lambda, alpha (IgA1 and
IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon and mu
constant region genes, as well as the myriad immunoglobulin
variable region genes. Full-length immunoglobulin "light chains"
(about 25 KDa or 214 amino acids) are encoded by a variable region
gene at the NH2-terminus (about 110 amino acids) and a kappa or
lambda constant region gene at the COOH--terminus. Full-length
immunoglobulin "heavy chains" (about 50 KDa or 446 amino acids),
are similarly encoded by a variable region gene (about 116 amino
acids) and one of the other aforementioned constant region genes,
e.g., gamma (encoding about 330 amino acids).
[0225] The term "antigen-binding fragment" of an antibody (or
simply "antibody portion," or "fragment"), as used herein, refers
to one or more fragments of a full-length antibody that retain the
ability to specifically bind to the antigen, e.g., 279 polypeptide
or fragment thereof. Examples of antigen-binding fragments of the
anti-279 antibody include, but are not limited to: (i) a Fab
fragment, a monovalent fragment consisting of the VL, VH, CL and
CH1 domains; (ii) a F(ab').sub.2 fragment, a bivalent fragment
comprising two Fab fragments linked by a disulfide bridge at the
hinge region; (iii) a Fd fragment consisting of the VH and CH1
domains; (iv) a Fv fragment consisting of the VL and VH domains of
a single arm of an antibody, (v) a dAb fragment (Ward et al.,
(1989) Nature 341:544-546), which consists of a VH domain; and (vi)
an isolated complementarity determining region (CDR). Furthermore,
although the two domains of the Fv fragment, VL and VH, are coded
for by separate genes, they can be joined, using recombinant
methods, by a synthetic linker that enables them to be made as a
single protein chain in which the VL and VH regions pair to form
monovalent molecules (known as single chain Fv (scFv); see e.g.,
Bird et al. (1988) Science 242:423-426; and Huston et al. (1988)
Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain
antibodies are also encompassed within the term "antigen-binding
fragment" of an antibody. These antibody fragments are obtained
using conventional techniques known to those with skill in the art,
and the fragments are screened for utility in the same manner as
are intact antibodies.
[0226] The anti-279 antibody can be a polyclonal or a monoclonal
antibody. In other embodiments, the antibody can be recombinantly
produced, e.g., produced by phage display or by combinatorial
methods.
[0227] Phage display and combinatorial methods for generating
anti-279 antibodies are known in the art (as described in, e.g.,
Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. International
Publication No. WO 92/18619; Dower et al. International Publication
No. WO 91/17271; Winter et al. International Publication WO
92/20791; Markland et al. International Publication No. WO
92/15679; Breitling et al. International Publication WO 93/01288;
McCafferty et al. International Publication No. WO 92/01047;
Garrard et al. International Publication No. WO 92/09690; Ladner et
al. International Publication No. WO 90/02809; Fuchs et al. (1991)
Bio/technology 9:1370-1372; Hay et al. (1992) Hum Antibod
Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;
Griffths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J
Mol Biol 226:889-896; Clackson et al. (1991) Nature 352:624-628;
Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991)
Biotechnology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res
19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982, the
contents of all of which are incorporated by reference herein).
[0228] In one embodiment, the anti-279 antibody is a fully human
antibody (e.g., an antibody made in a mouse which has been
genetically engineered to produce an antibody from a human
immunoglobulin sequence), or a non-human antibody, e.g., a rodent
(mouse or rat), goat, primate (e.g., monkey), camel antibody.
Preferably, the non-human antibody is a rodent (mouse or rat
antibody). Method of producing rodent antibodies are known in the
art.
[0229] Human monoclonal antibodies can be generated using
transgenic mice carrying the human immunoglobulin genes rather than
the mouse system. Splenocytes from these transgenic mice immunized
with the antigen of interest are used to produce hybridomas that
secrete human mAbs with specific affinities for epitopes from a
human protein (see, e.g., Wood et al. International Application WO
91/00906, Kucherlapati et al. PCT publication WO 91/10741; Lonberg
et al. International Application WO 92/03918; Kay et al.
International Application 92/03917; Lonberg, N. et al. 1994 Nature
368:856-859; Green, L. L. et al. 1994 Nature Genet. 7:13-21;
Morrison, S.L. et al. 1994 Proc. Natl. Acad. Sci. USA 81:6851-6855;
Bruggeman et al. 1993 Year Immunol 7:33-40; Tuaillon et al. 1993
PNAS 90:3720-3724; Bruggeman et al. 1991 Eur J Immunol
21:1323-1326).
[0230] An anti-279 antibody can be one in which the variable
region, or a portion thereof, e.g., the CDR's, are generated in a
non-human organism, e.g., a rat or mouse. Chimeric, CDR-grafted,
and humanized antibodies are within the invention. Antibodies
generated in a non-human organism, e.g., a rat or mouse, and then
modified, e.g., in the variable framework or constant region, to
decrease antigenicity in a human are within the invention.
[0231] Chimeric antibodies can be produced by recombinant DNA
techniques known in the art. For example, a gene encoding the Fe
constant region of a murine (or other species) monoclonal antibody
molecule is digested with restriction enzymes to remove the region
encoding the murine Fc, and the equivalent portion of a gene
encoding a human Fc constant region is substituted (see Robinson et
al., International Patent Publication PCT/US86/02269; Akira, et
al., European Patent Application 184,187; Taniguchi, M., European
Patent Application 171,496; Morrison et al., European Patent
Application 173,494; Neuberger et al., International Application WO
86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al.,
European Patent Application 125,023; Better et al. (1988 Science
240:1041-1043); Liu et al. (1987) PNAS 84:3439-3443; Liu et al.,
1987, J. Immunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214-218;
Nishimura et al., 1987, Canc. Res. 47:999-1005; Wood et al. (1985)
Nature 314:446-449; and Shaw et al., 1988, J. Natl Cancer Inst.
80:1553-1559).
[0232] A humanized or CDR-grafted antibody will have at least one
or two but generally all three recipient CDR's (of heavy and or
light immuoglobulin chains) replaced with a donor CDR. The antibody
may be replaced with at least a portion of a non-human CDR or only
some of the CDR's may be replaced with non-human CDR's. It is only
necessary to replace the number of CDR's required for binding of
the humanized antibody to a 279 or a fragment thereof. Preferably,
the donor will be a rodent antibody, e.g., a rat or mouse antibody,
and the recipient will be a human framework or a human consensus
framework. Typically, the immunoglobulin providing the CDR's is
called the "donor" and the immunoglobulin providing the framework
is called the "acceptor." In one embodiment, the donor
immunoglobulin is a non-human (e.g., rodent). The acceptor
framework is a naturally-occurring (e.g., a human) framework or a
consensus framework, or a sequence about 85% or higher, preferably
90%, 95%, 99% or higher identical thereto.
[0233] As used herein, the term "consensus sequence" refers to the
sequence formed from the most frequently occurring amino acids (or
nucleotides) in a family of related sequences (See e.g., Winnaker,
From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987).
In a family of proteins, each position in the consensus sequence is
occupied by the amino acid occurring most frequently at that
position in the family. If two amino acids occur equally
frequently, either can be included in the consensus sequence. A
"consensus framework" refers to the framework region in the
consensus immunoglobulin sequence.
[0234] An antibody can be humanized by methods known in the art.
Humanized antibodies can be generated by replacing sequences of the
Fv variable region which are not directly involved in antigen
binding with equivalent sequences from human Fv variable regions.
General methods for generating humanized antibodies are provided by
Morrison, S. L., 1985, Science 229:1202-1207, by Oi et al., 1986,
BioTechniques 4:214, and by Queen et al. U.S. Pat. Nos. 5,585,089,
5,693,761 and 5,693,762, the contents of all of which are hereby
incorporated by reference. Those methods include isolating,
manipulating, and expressing the nucleic acid sequences that encode
all or part of immunoglobulin Fv variable regions from at least one
of a heavy or light chain. Sources of such nucleic acid are well
known to those skilled in the art and, for example, may be obtained
from a hybridoma producing an antibody against a 279 polypeptide or
fragment thereof. The recombinant DNA encoding the humanized
antibody, or fragment thereof, can then be cloned into an
appropriate expression vector.
[0235] Humanized or CDR-grafted antibodies can be produced by
CDR-grafting or CDR substitution, wherein one, two, or all CDR's of
an immunoglobulin chain can be replaced. See e.g., U.S. Pat. No.
5,225,539; Jones et al. 1986 Nature 321:552-525; Verhoeyan et al.
1988 Science 239:1534; Beidler et al. 1988 J. Immunol.
141:4053-4060; Winter U.S. Pat. No. 5,225,539, the contents of all
of which are hereby expressly incorporated by reference. Winter
describes a CDR-grafting method which may be used to prepare the
humanized antibodies of the present invention (UK Patent
Application GB 2188638A, filed on Mar. 26, 1987; Winter U.S. Pat.
No. 5,225,539), the contents of which is expressly incorporated by
reference.
[0236] Also within the scope of the invention are humanized
antibodies in which specific amino acids have been substituted,
deleted or added. Preferred humanized antibodies have amino acid
substitutions in the framework region, such as to improve binding
to the antigen. For example, a humanized antibody will have
framework residues identical to the donor framework residue or to
another amino acid other than the recipient framework residue. To
generate such antibodies, a selected, small number of acceptor
framework residues of the humanized immunoglobulin chain can be
replaced by the corresponding donor amino acids. Preferred
locations of the substitutions include amino acid residues adjacent
to the CDR, or which are capable of interacting with a CDR (see
e.g., U.S. Pat. No. 5,585,089). Criteria for selecting amino acids
from the donor are described in U.S. Pat. No. 5,585,089, e.g.,
columns 12-16 of U.S. Pat. No. 5,585,089, the e.g., columns 12-16
of U.S. Pat. No. 5,585,089, the contents of which are hereby
incorporated by reference. Other techniques for humanizing
antibodies are described in Padlan et al. EP 519596 A1, published
on Dec. 23, 1992.
[0237] In preferred embodiments an antibody can be made by
immunizing with purified 279 antigen, or a fragment thereof, e.g.,
a fragment comprising a GPCR domain, membrane associated antigen,
tissue, e.g., crude tissue preparations, whole cells, preferably
living cells, lysed cells, or cell fractions, e.g., membrane
fractions.
[0238] A full-length 279 protein or, antigenic peptide fragment of
279 can be used as an immunogen or can be used to identify anti-279
antibodies made with other immunogens, e.g., cells, membrane
preparations, and the like. The antigenic peptide of 279 should
include at least 8 amino acid residues of the amino acid sequence
shown in SEQ ID NO: 2 and encompasses an epitope of 279.
Preferably, the antigenic peptide includes at least 10 amino acid
residues, more preferably at least 15 amino acid residues, even
more preferably at least 20 amino acid residues, and most
preferably at least 30 amino acid residues.
[0239] Fragments of 279 which include residues about about 44 to
51, about 75 to 91, about 114 to 133, about 155 to 185, about 206
to 224, or about 246 to 271 SEQ ID NO: 2 can be used to make, e.g.,
used as immunogens or used to characterize the specificity of an
antibody, antibodies against hydrophilic regions of the 279
protein. Similarly, fragments of 279 which include residues about
23 to 43, about 52 to 74, about 92 to 113, about 134 to 154, about
186 to about 205, about 225 to 245, or about 272 to 289 of SEQ ID
NO: 2 can be used to make an antibody against a hydrophobic region
of the 279 protein; fragments of 279 which include residues about 1
to 22, 75 to 91, or about 155 to 185, or about 246 to 271 of SEQ ID
NO: 2 can be used to make an antibody against an extracellular
region of the 279 protein; fragments of 279 which include residues
about 44 to 51, about 114 to 133, about 206 to 224, or about 290 to
362 of SEQ ID NO: 2 can be used to make an antibody against an
intracellular region of the 279 protein; a fragment of 279 which
include residues about 34 to 286 (or a fragment thereof, e.g., 34
to 50, 50 to 100, 100 to 150, 150 to 200, 200 to 250, or 250 to
286) of SEQ ID NO: 2 can be used to make an antibody against the
GPCR region of the 279 protein.
[0240] Antibodies reactive with, or specific for, any of these
regions, or other regions or domains described herein are
provided.
[0241] Antibodies which bind only native 279 protein, only
denatured or otherwise non-native 279 protein, or which bind both,
are with in the invention. Antibodies with linear or conformational
epitopes are within the invention. Conformational epitopes can
sometimes be identified by identifying antibodies which bind to
native but not denatured 279 protein.
[0242] Preferred epitopes encompassed by the antigenic peptide are
regions of 279 are located on the surface of the protein, e.g.,
hydrophilic regions, as well as regions with high antigenicity. For
example, an Emini surface probability analysis of the human 279
protein sequence can be used to indicate the regions that have a
particularly high probability of being localized to the surface of
the 279 protein and are thus likely to constitute surface residues
useful for targeting antibody production.
[0243] In a preferred embodiment the antibody can bind to the
extracellular portion of the 279 protein, e.g., it can bind to a
whole cell that expresses the 279 protein. In another embodiment,
the antibody binds an intracellular portion of the 279 protein. In
preferred embodiments antibodies can bind one or more of purified
antigen, membrane associated antigen, tissue, e.g., tissue
sections, whole cells, preferably living cells, lysed cells, cell
fractions, e.g., membrane fractions.
[0244] The anti-279 antibody can be a single chain antibody. A
single-chain antibody (scFV) may be engineered (see, for example,
Colcher, D. et al. (1999) Ann N Y Acad Sci 880:263-80; and Reiter,
Y. (1996) Clin Cancer Res 2:245-52). The single chain antibody can
be dimerized or multimerized to generate multivalent antibodies
having specificities for different epitopes of the same target 279
protein.
[0245] In a preferred embodiment the antibody has effector function
and/or can fix complement. In other embodiments the antibody does
not recruit effector cells; or fix complement.
[0246] In a preferred embodiment, the antibody has reduced or no
ability to bind an Fc receptor. For example, it is a isotype or
subtype, fragment or other mutant, which does not support binding
to an Fe receptor, e.g., it has a mutagenized or deleted Fc
receptor binding region.
[0247] In a preferred embodiment, an anti-279 antibody alters
(e.g., increases or decreases) the sensing of environmental
stimuli, e.g., small molecules; sensing of biological messengers,
e.g., secreted hormones; or signaling to G proteins of a 279
polypeptide. For example, the antibody can bind at or in proximity
to the active site, e.g., to an epitope that includes a residue
located from about 114 to 116 of SEQ ID NO: 2
[0248] The antibody can be coupled to a toxin, e.g., a polypeptide
toxin, e,g, ricin or diphtheria toxin or active fragment hereof, or
a radioactive nucleus, or imaging agent, e.g. a radioactive,
enzymatic, or other, e.g., imaging agent, e.g., a NMR contrast
agent. Labels which produce detectable radioactive emissions or
fluorescence are preferred.
[0249] An anti-279 antibody (e.g., monoclonal antibody) can be used
to isolate 279 by standard techniques, such as affinity
chromatography or immunoprecipitation. Moreover, an anti-279
antibody can be used to detect 279 protein (e.g., in a cellular
lysate or cell supernatant) in order to evaluate the abundance and
pattern of expression of the protein. Anti-279 antibodies can be
used diagnostically to monitor protein levels in tissue as part of
a clinical testing procedure, e.g., to determine the efficacy of a
given treatment regimen. Detection can be facilitated by coupling
(i.e., physically linking) the antibody to a detectable substance
(i.e., antibody labelling). Examples of detectable substances
include various enzymes, prosthetic groups, fluorescent materials,
luminescent materials, bioluminescent materials, and radioactive
materials. Examples of suitable enzymes include horseradish
peroxidase, alkaline phosphatase, .beta.-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin, and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S or .sup.3H.
[0250] The invention also includes a nucleic acid which encodes an
anti-279 antibody, e.g., an anti-279 antibody described herein.
Also included are vectors which include the nucleic acid and cells
transformed with the nucleic acid, particularly cells which are
useful for producing an antibody, e.g., mammalian cells, e.g. CHO
or lymphatic cells.
[0251] The invention also includes cell lines, e.g., hybridomas,
which make an anti-279 antibody, e.g., and antibody described
herein, and method of using said cells to make a 279 antibody.
[0252] Recombinant Expression Vectors, Host Cells and Genetically
Engineered Cells
[0253] In another aspect, the invention includes, vectors,
preferably expression vectors, containing a nucleic acid encoding a
polypeptide described herein. As used herein, the term "vector"
refers to a nucleic acid molecule capable of transporting another
nucleic acid to which it has been linked and can include a plasmid,
cosmid or viral vector. The vector can be capable of autonomous
replication or it can integrate into a host DNA. Viral vectors
include, e.g., replication defective retroviruses, adenoviruses and
adeno-associated viruses.
[0254] A vector can include a 279 nucleic acid in a form suitable
for expression of the nucleic acid in a host cell. Preferably the
recombinant expression vector includes one or more regulatory
sequences operatively linked to the nucleic acid sequence to be
expressed. The term "regulatory sequence" includes promoters,
enhancers and other expression control elements (e.g.,
polyadenylation signals). Regulatory sequences include those which
direct constitutive expression of a nucleotide sequence, as well as
tissue-specific regulatory and/or inducible sequences. The design
of the expression vector can depend on such factors as the choice
of the host cell to be transformed, the level of expression of
protein desired, and the like. The expression vectors of the
invention can be introduced into host cells to thereby produce
proteins or polypeptides, including fusion proteins or
polypeptides, encoded by nucleic acids as described herein (e.g.,
279 proteins, mutant forms of 279 proteins, fusion proteins, and
the like).
[0255] The recombinant expression vectors of the invention can be
designed for expression of 279 proteins in prokaryotic or
eukaryotic cells. For example, polypeptides of the invention can be
expressed in E. coli, insect cells (e.g., using baculovirus
expression vectors), yeast cells or mammalian cells. Suitable host
cells are discussed further in Goeddel, (1990) Gene Expression
Technology: Methods in Enzymology 185, Academic Press, San Diego,
Calif. Alternatively, the recombinant expression vector can be
transcribed and translated in vitro, for example using T7 promoter
regulatory sequences and T7 polymerase.
[0256] Expression of proteins in prokaryotes is most often carried
out in E. coli with vectors containing constitutive or inducible
promoters directing the expression of either fusion or non-fusion
proteins. Fusion vectors add a number of amino acids to a protein
encoded therein, usually to the amino terminus of the recombinant
protein. Such fusion vectors typically serve three purposes: 1) to
increase expression of recombinant protein; 2) to increase the
solubility of the recombinant protein; and 3) to aid in the
purification of the recombinant protein by acting as a ligand in
affinity purification. Often, a proteolytic cleavage site is
introduced at the junction of the fusion moiety and the recombinant
protein to enable separation of the recombinant protein from the
fusion moiety subsequent to purification of the fusion protein.
Such enzymes, and their cognate recognition sequences, include
Factor Xa, thrombin and enterokinase. Typical fusion expression
vectors include pGEX (Pharmacia Biotech Inc; Smith, D. B. and
Johnson, K. S. (1988) Gene 67:31-40), pMAL (New England Biolabs,
Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse
glutathione S-transferase (GST), maltose E binding protein, or
protein A, respectively, to the target recombinant protein.
[0257] Purified fusion proteins can be used in 279 activity assays,
(e.g., direct assays or competitive assays described in detail
below), or to generate antibodies specific for 279 proteins. In a
preferred embodiment, a fusion protein expressed in a retroviral
expression vector of the present invention can be used to infect
bone marrow cells which are subsequently transplanted into
irradiated recipients. The pathology of the subject recipient is
then examined after sufficient time has passed (e.g., six
weeks).
[0258] To maximize recombinant protein expression in E. coli is to
express the protein in a host bacteria with an impaired capacity to
proteolytically cleave the recombinant protein (Gottesman, S.,
(1990) Gene Expression Technology: Methods in Enzymology 185,
Academic Press, San Diego, Calif. 119-128). Another strategy is to
alter the nucleic acid sequence of the nucleic acid to be inserted
into an expression vector so that the individual codons for each
amino acid are those preferentially utilized in E. coli (Wada et
al., (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of
nucleic acid sequences of the invention can be carried out by
standard DNA synthesis techniques.
[0259] The 279 expression vector can be a yeast expression vector,
a vector for expression in insect cells, e.g., a baculovirus
expression vector or a vector suitable for expression in mammalian
cells.
[0260] When used in mammalian cells, the expression vector's
control functions can be provided by viral regulatory elements. For
example, commonly used promoters are derived from polyoma,
Adenovirus 2, cytomegalovirus and Simian Virus 40.
[0261] In another embodiment, the promoter is an inducible
promoter, e.g., a promoter regulated by a steroid hormone, by a
polypeptide hormone (e.g., by means of a signal transduction
pathway), or by a heterologous polypeptide (e.g., the
tetracycline-inducible systems, "Tet-On" and "Tet-Off"; see, e.g.,
Clontech Inc., CA, Gossen and Bujard (1992) Proc. Natl. Acad. Sci.
USA 89:5547, and Paillard (1989) Human Gene Therapy 9:983).
[0262] In another embodiment, the recombinant mammalian expression
vector is capable of directing expression of the nucleic acid
preferentially in a particular cell type (e.g., tissue-specific
regulatory elements are used to express the nucleic acid).
Non-limiting examples of suitable tissue-specific promoters include
the albumin promoter (liver-specific; Pinkert et al. (1987) Genes
Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton
(1988) Adv. Immunol. 43:235-275), in particular promoters of T cell
receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and
immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and
Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g.,
the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl.
Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund
et al. (1985) Science 230:912-916), and mammary gland-specific
promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and
European Application Publication No. 264,166).
Developmentally-regulated promoters are also encompassed, for
example, the murine hox promoters (Kessel and Gruss (1990) Science
249:374-379) and the .alpha.-fetoprotein promoter (Campes and
Tilghman (1989) Genes Dev. 3:537-546).
[0263] The invention further provides a recombinant expression
vector comprising a DNA molecule of the invention cloned into the
expression vector in an antisense orientation. Regulatory sequences
(e.g., viral promoters and/or enhancers) operatively linked to a
nucleic acid cloned in the antisense orientation can be chosen
which direct the constitutive, tissue specific or cell type
specific expression of antisense RNA in a variety of cell types.
The antisense expression vector can be in the form of a recombinant
plasmid, phagemid or attenuated virus.
[0264] Another aspect the invention provides a host cell which
includes a nucleic acid molecule described herein, e.g., a 279
nucleic acid molecule within a recombinant expression vector or a
279 nucleic acid molecule containing sequences which allow it to
homologously recombine into a specific site of the host cell's
genome. The terms "host cell" and "recombinant host cell" are used
interchangeably herein. Such terms refer not only to the particular
subject cell but to the progeny or potential progeny of such a
cell. Because certain modifications may occur in succeeding
generations due to either mutation or environmental influences,
such progeny may not, in fact, be identical to the parent cell, but
are still included within the scope of the term as used herein.
[0265] A host cell can be any prokaryotic or eukaryotic cell. For
example, a 279 protein can be expressed in bacterial cells (such as
E. coli), insect cells, yeast or mammalian cells (such as Chinese
hamster ovary cells (CHO) or COS cells (African green monkey kidney
cells CV-1 origin SV40 cells; Gluzman (1981) CellI23:175-182)).
Other suitable host cells are known to those skilled in the
art.
[0266] Vector DNA can be introduced into host cells via
conventional transformation or transfection techniques. As used
herein, the terms "transformation" and "transfection" are intended
to refer to a variety of art-recognized techniques for introducing
foreign nucleic acid (e.g., DNA) into a host cell, including
calcium phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, lipofection, or
electroporation.
[0267] A host cell of the invention can be used to produce (i.e.,
express) a 279 protein. Accordingly, the invention further provides
methods for producing a 279 protein using the host cells of the
invention. In one embodiment, the method includes culturing the
host cell of the invention (into which a recombinant expression
vector encoding a 279 protein has been introduced) in a suitable
medium such that a 279 protein is produced. In another embodiment,
the method further includes isolating a 279 protein from the medium
or the host cell.
[0268] In another aspect, the invention features, a cell or
purified preparation of cells which include a 279 transgene, or
which otherwise misexpress 279. The cell preparation can consist of
human or non-human cells, e.g., rodent cells, e.g., mouse or rat
cells, rabbit cells, or pig cells. In preferred embodiments, the
cell or cells include a 279 transgene, e.g., a heterologous form of
a 279, e.g., a gene derived from humans (in the case of a non-human
cell). The 279 transgene can be misexpressed, e.g., overexpressed
or underexpressed. In other preferred embodiments, the cell or
cells include a gene that mis-expresses an endogenous 279, e.g., a
gene the expression of which is disrupted, e.g., a knockout. Such
cells can serve as a model for studying disorders that are related
to mutated or mis-expressed 279 alleles or for use in drug
screening.
[0269] In another aspect, the invention features, a human cell,
e.g., a hematopoietic stem cell, transformed with nucleic acid
which encodes a subject 279 polypeptide.
[0270] Also provided are cells, preferably human cells, e.g., human
hematopoietic or fibroblast cells, in which an endogenous 279 is
under the control of a regulatory sequence that does not normally
control the expression of the endogenous 279 gene. The expression
characteristics of an endogenous gene within a cell, e.g., a cell
line or microorganism, can be modified by inserting a heterologous
DNA regulatory element into the genome of the cell such that the
inserted regulatory element is operably linked to the endogenous
279 gene. For example, an endogenous 279 gene which is
"transcriptionally silent," e.g., not normally expressed, or
expressed only at very low levels, may be activated by inserting a
regulatory element which is capable of promoting the expression of
a normally expressed gene product in that cell. Techniques such as
targeted homologous recombinations, can be used to insert the
heterologous DNA as described in, e.g., Chappel, U.S. Pat. No.
5,272,071; WO 91/06667, published in May 16, 1991.
[0271] In a preferred embodiment, recombinant cells described
herein can be used for replacement therapy in a subject. For
example, a nucleic acid encoding a 279 polypeptide operably linked
to an inducible promoter (e.g., a steroid hormone
receptor-regulated promoter) is introduced into a human or
nonhuman, e.g., mammalian, e.g., porcine recombinant cell. The cell
is cultivated and encapsulated in a biocompatible material, such as
poly-lysine alginate, and subsequently implanted into the subject.
See, e.g., Lanza (1996) Nat. Biotechnol. 14:1107; Joki et al.
(2001) Nat. Biotechnol. 19:35; and U.S. Pat. No. 5,876,742.
Production of 279 polypeptide can be regulated in the subject by
administering an agent (e.g., a steroid hormone) to the subject. In
another preferred embodiment, the implanted recombinant cells
express and secrete an antibody specific for a 279 polypeptide. The
antibody can be any antibody or any antibody derivative described
herein.
[0272] Transgenic Animals
[0273] The invention provides non-human transgenic animals. Such
animals are useful for studying the function and/or activity of a
279 protein and for identifying and/or evaluating modulators of 279
activity. As used herein, a "transgenic animal" is a non-human
animal, preferably a mammal, more preferably a rodent such as a rat
or mouse, in which one or more of the cells of the animal includes
a transgene. Other examples of transgenic animals include non-human
primates, sheep, dogs, cows, goats, chickens, amphibians, and the
like. A transgene is exogenous DNA or a rearrangement, e.g., a
deletion of endogenous chromosomal DNA, which preferably is
integrated into or occurs in the genome of the cells of a
transgenic animal. A transgene can direct the expression of an
encoded gene product in one or more cell types or tissues of the
transgenic animal, other transgenes, e.g., a knockout, reduce
expression. Thus, a transgenic animal can be one in which an
endogenous 279 gene has been altered by, e.g., by homologous
recombination between the endogenous gene and an exogenous DNA
molecule introduced into a cell of the animal, e.g., an embryonic
cell of the animal, prior to development of the animal.
[0274] Intronic sequences and polyadenylation signals can also be
included in the transgene to increase the efficiency of expression
of the transgene. A tissue-specific regulatory sequence(s) can be
operably linked to a transgene of the invention to direct
expression of a 279 protein to particular cells. A transgenic
founder animal can be identified based upon the presence of a 279
transgene in its genome and/or expression of 279 mRNA in tissues or
cells of the animals. A transgenic founder animal can then be used
to breed additional animals carrying the transgene. Moreover,
transgenic animals carrying a transgene encoding a 279 protein can
further be bred to other transgenic animals carrying other
transgenes.
[0275] 279 proteins or polypeptides can be expressed in transgenic
animals or plants, e.g., a nucleic acid encoding the protein or
polypeptide can be introduced into the genome of an animal. In
preferred embodiments the nucleic acid is placed under the control
of a tissue specific promoter, e.g., a milk or egg specific
promoter, and recovered from the milk or eggs produced by the
animal. Suitable animals are mice, pigs, cows, goats, and
sheep.
[0276] The invention also includes a population of cells from a
transgenic animal, as discussed, e.g., below.
[0277] Uses
[0278] The nucleic acid molecules, proteins, protein homologues,
and antibodies described herein can be used in one or more of the
following methods: a) screening assays; b) predictive medicine
(e.g., diagnostic assays, prognostic assays, monitoring clinical
trials, and pharmacogenetics); and c) methods of treatment (e.g.,
therapeutic and prophylactic).
[0279] The isolated nucleic acid molecules of the invention can be
used, for example, to express a 279 protein (e.g., via a
recombinant expression vector in a host cell in gene therapy
applications), to detect a 279 mRNA (e.g., in a biological sample)
or a genetic alteration in a 279 gene, and to modulate 279
activity, as described further below. The 279 proteins can be used
to treat disorders characterized by insufficient or excessive
production of a 279 substrate or production of 279 inhibitors. In
addition, the 279 proteins can be used to screen for naturally
occurring 279 substrates, to screen for drugs or compounds which
modulate 279 activity, as well as to treat disorders characterized
by insufficient or excessive production of 279 protein or
production of 279 protein forms which have decreased, aberrant or
unwanted activity compared to 279 wild type protein (e.g.,
angiogenesis related disorders such as cancer, cardiovascular
disorders such as atherosclerosis, and disorders involving vascular
tone). Moreover, the anti-279 antibodies of the invention can be
used to detect and isolate 279 proteins, regulate the
bioavailability of 279 proteins, and modulate 279 activity.
[0280] A method of evaluating a compound for the ability to
interact with, e.g., bind, a subject 279 polypeptide is provided.
The method includes: contacting the compound with the subject 279
polypeptide; and evaluating ability of the compound to interact
with, e.g., to bind or form a complex with the subject 279
polypeptide. This method can be performed in vitro, e.g., in a cell
free system, or in vivo, e.g., in a two-hybrid interaction trap
assay. This method can be used to identify naturally occurring
molecules that interact with subject 279 polypeptide. It can also
be used to find natural or synthetic inhibitors of subject 279
polypeptide. Screening methods are discussed in more detail
below.
[0281] Screening Assays
[0282] The invention provides methods (also referred to herein as
"screening assays") for identifying modulators, i.e., candidate or
test compounds or agents (e.g., proteins, peptides,
peptidomimetics, peptoids, small molecules or other drugs) which
bind to 279 proteins, have a stimulatory or inhibitory effect on,
for example, 279 expression or 279 activity, or have a stimulatory
or inhibitory effect on, for example, the expression or activity of
a 279 substrate. Compounds thus identified can be used to modulate
the activity of target gene products (e.g., 279 genes) in a
therapeutic protocol, to elaborate the biological function of the
target gene product, or to identify compounds that disrupt normal
target gene interactions.
[0283] In one embodiment, the invention provides assays for
screening candidate or test compounds which are substrates of a 279
protein or polypeptide or a biologically active portion thereof. In
another embodiment, the invention provides assays for screening
candidate or test compounds that bind to or modulate an activity of
a 279 protein or polypeptide or a biologically active portion
thereof.
[0284] The 279 polypeptide can be tagged as described herein,
purified or partially purified and assayed for signaling to G
proteins in a variety of assay conditions. Samples which show no
activity at 0.1 mM protein can be considered inactive under those
conditions. Assay conditions such as buffer, pH, and substrate, can
be permuted until an appropriate one is found (e.g. pH 6.0 to 9.0,
including or not including calcium ions).
[0285] Assaying a 279 protein activity in the presence of
inhibitors can be used to determine the nature of the signaling
activity. The assay can include additional controls and steps to
insure that the observed activity results from a 279 polypeptide.
For example, control samples, e.g., samples produced from cells
transformed with a control vector instead of a vector
overexpressing a 279 nucleic acid, can be tested in parallel. The
activity can also be tracked in chromatography fractions, e.g., to
determine if the activity and the 279 polypeptide co-purify.
Additional assays and conditions are described, e.g., in Yamaoka et
al. (1998) J Biol Chem 273:11895-11901, and Thien-Khai et al.
(1997) J Biol Chem 272:31315-31320.
[0286] The test compounds of the present invention can be obtained
using any of the numerous approaches in combinatorial library
methods known in the art, including: biological libraries; peptoid
libraries (libraries of molecules having the functionalities of
peptides, but with a novel, non-peptide backbone which are
resistant to enzymatic degradation but which nevertheless remain
bioactive; see, e.g., Zuckermann, R. N. et al. (1994) J. Med. Chem.
37:2678-85); spatially addressable parallel solid phase or solution
phase libraries; synthetic library methods requiring deconvolution;
the `one-bead one-compound` library method; and synthetic library
methods using affinity chromatography selection. The biological
library and peptoid library approaches are limited to peptide
libraries, while the other four approaches are applicable to
peptide, non-peptide oligomer or small molecule libraries of
compounds (Lam (1997) Anticancer Drug Des. 12:145).
[0287] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example in: DeWitt et al. (1993) Proc.
Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl.
Acad. Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem.
37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994)
Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew.
Chem. Int. Ed. Engl. 33:2061; and Gallop et al. (1994) J. Med.
Chem. 37:1233.
[0288] Libraries of compounds may be presented in solution (e.g.,
Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991)
Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556),
bacteria (Ladner, U.S. Pat. No. 5,223,409), spores (Ladner U.S.
Pat. No. 5,223,409), plasmids (Cull et al. (1992) Proc Natl Acad
Sci USA 89:1865-1869) or on phage (Scott and Smith (1990) Science
249:386-390; Devlin (1990) Science 249:404-406; Cwirla et al.
(1990) Proc. Natl. Acad. Sci. 87:6378-6382; Felici (1991) J. Mol.
Biol. 222:301-310; Ladner supra.).
[0289] In one embodiment, an assay is a cell-based assay in which a
cell which expresses a 279 protein or biologically active portion
thereof is contacted with a test compound, and the ability of the
test compound to modulate 279 activity is determined. Determining
the ability of the test compound to modulate 279 activity can be
accomplished by monitoring, for example, sensing environmental
stimuli, e.g., a GPCR ligand, a small molecules; sensing biological
messengers, e.g., secreted hormones; or signaling to G proteins.
The cell, for example, can be of mammalian origin, e.g., human.
[0290] The ability of the test compound to modulate 279 binding to
a compound, e.g., a 279 substrate, or to bind to 279 can also be
evaluated. This can be accomplished, for example, by coupling the
compound, e.g., the substrate, with a radioisotope or enzymatic
label such that binding of the compound, e.g., the substrate, to
279 can be determined by detecting the labeled compound, e.g.,
substrate, in a complex. Alternatively, 279 could be coupled with a
radioisotope or enzymatic label to monitor the ability of a test
compound to modulate 279 binding to a 279 substrate in a complex.
For example, compounds (e.g., 279 substrates) can be labeled with
.sup.125I, .sup.35S, .sup.14C, or .sup.3H, either directly or
indirectly, and the radioisotope detected by direct counting of
radioemmission or by scintillation counting. Alternatively,
compounds can be enzymatically labeled with, for example,
horseradish peroxidase, alkaline phosphatase, or luciferase, and
the enzymatic label detected by determination of conversion of an
appropriate substrate to product.
[0291] The ability of a compound (e.g., a 279 substrate) to
interact with 279 with or without the labeling of any of the
interactants can be evaluated. For example, a microphysiometer can
be used to detect the interaction of a compound with 279 without
the labeling of either the compound or the 279. McConnell, H. M. et
al. (1992) Science 257:1906-1912. As used herein, a
"microphysiometer" (e.g., Cytosensor) is an analytical instrument
that measures the rate at which a cell acidifies its environment
using a light-addressable potentiometric sensor (LAPS). Changes in
this acidification rate can be used as an indicator of the
interaction between a compound and 279.
[0292] In yet another embodiment, a cell-free assay is provided in
which a 279 protein or biologically active portion thereof is
contacted with a test compound and the ability of the test compound
to bind to the 279 protein or biologically active portion thereof
is evaluated. Preferred biologically active portions of the 279
proteins to be used in assays of the present invention include
fragments which participate in interactions with non-279 molecules,
e.g., fragments with high surface probability scores.
[0293] Soluble and/or membrane-bound forms of isolated proteins
(e.g., 279 proteins or biologically active portions thereof) can be
used in the cell-free assays of the invention. When membrane-bound
forms of the protein are used, it may be desirable to utilize a
solubilizing agent. Examples of such solubilizing agents include
non-ionic detergents such as n-octylglucoside, n-dodecylglucoside,
n-dodecylmaltoside, octanoyl-N-methylglucamide,
decanoyl-N-methylglucamide, Triton.RTM. X-100, Triton.RTM. X-114,
Thesit.RTM., Isotridecypoly(ethylene glycol ether).sub.n,
3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),
3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane
sulfonate (CHAPSO), or
N-dodecyl.dbd.N,N-dimethyl-3-ammonio-1-propane sulfonate.
[0294] Cell-free assays involve preparing a reaction mixture of the
target gene protein and the test compound under conditions and for
a time sufficient to allow the two components to interact and bind,
thus forming a complex that can be removed and/or detected.
[0295] The interaction between two molecules can also be detected,
e.g., using fluorescence energy transfer (FET) (see, for example,
Lakowicz et al., U.S. Pat. No. 5,631,169; Stavrianopoulos, et al.,
U.S. Pat. No. 4,868,103). A fluorophore label on the first, `donor`
molecule is selected such that its emitted fluorescent energy will
be absorbed by a fluorescent label on a second, `acceptor`
molecule, which in turn is able to fluoresce due to the absorbed
energy. Alternately, the `donor` protein molecule may simply
utilize the natural fluorescent energy of tryptophan residues.
Labels are chosen that emit different wavelengths of light, such
that the `acceptor` molecule label may be differentiated from that
of the `donor`. Since the efficiency of energy transfer between the
labels is related to the distance separating the molecules, the
spatial relationship between the molecules can be assessed. In a
situation in which binding occurs between the molecules, the
fluorescent emission of the `acceptor` molecule label in the assay
should be maximal. An FET binding event can be conveniently
measured through standard fluorometric detection means well known
in the art (e.g., using a fluorimeter).
[0296] In another embodiment, determining the ability of the 279
protein to bind to a target molecule can be accomplished using
real-time Biomolecular Interaction Analysis (BIA) (see, e.g.,
Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345
and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705).
"Surface plasmon resonance" or "BIA" detects biospecific
interactions in real time, without labeling any of the interactants
(e.g., BlAcore). Changes in the mass at the binding surface
(indicative of a binding event) result in alterations of the
refractive index of light near the surface (the optical phenomenon
of surface plasmon resonance (SPR)), resulting in a detectable
signal which can be used as an indication of real-time reactions
between biological molecules.
[0297] In one embodiment, the target gene product or the test
substance is anchored onto a solid phase. The target gene
product/test compound complexes anchored on the solid phase can be
detected at the end of the reaction. Preferably, the target gene
product can be anchored onto a solid surface, and the test
compound, (which is not anchored), can be labeled, either directly
or indirectly, with detectable labels discussed herein.
[0298] It may be desirable to immobilize either 279, an anti-279
antibody or its target molecule to facilitate separation of
complexed from uncomplexed forms of one or both of the proteins, as
well as to accommodate automation of the assay. Binding of a test
compound to a 279 protein, or interaction of a 279 protein with a
target molecule in the presence and absence of a candidate
compound, can be accomplished in any vessel suitable for containing
the reactants. Examples of such vessels include microtiter plates,
test tubes, and micro-centrifuge tubes. In one embodiment, a fusion
protein can be provided which adds a domain that allows one or both
of the proteins to be bound to a matrix. For example,
glutathione-S-transferase/279 fusion proteins or
glutathione-S-transferas- e/target fusion proteins can be adsorbed
onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.)
or glutathione derivatized microtiter plates, which are then
combined with the test compound or the test compound and either the
non-adsorbed target protein or 279 protein, and the mixture
incubated under conditions conducive to complex formation (e.g., at
physiological conditions for salt and pH). Following incubation,
the beads or microtiter plate wells are washed to remove any
unbound components, the matrix immobilized in the case of beads,
complex determined either directly or indirectly, for example, as
described above. Alternatively, the complexes can be dissociated
from the matrix, and the level of 279 binding or activity
determined using standard techniques.
[0299] Other techniques for immobilizing either a 279 protein or a
target molecule on matrices include using conjugation of biotin and
streptavidin. Biotinylated 279 protein or target molecules can be
prepared from biotin-NHS (N-hydroxy-succinimide) using techniques
known in the art (e.g., biotinylation kit, Pierce Chemicals,
Rockford, Ill.), and immobilized in the wells of
streptavidin-coated 96 well plates (Pierce Chemical).
[0300] In order to conduct the assay, the non-immobilized component
is added to the coated surface containing the anchored component.
After the reaction is complete, unreacted components are removed
(e.g., by washing) under conditions such that any complexes formed
will remain immobilized on the solid surface. The detection of
complexes anchored on the solid surface can be accomplished in a
number of ways. Where the previously non-immobilized component is
pre-labeled, the detection of label immobilized on the surface
indicates that complexes were formed. Where the previously
non-immobilized component is not pre-labeled, an indirect label can
be used to detect complexes anchored on the surface; e.g., using a
labeled antibody specific for the immobilized component (the
antibody, in turn, can be directly labeled or indirectly labeled
with, e.g., a labeled anti-Ig antibody).
[0301] In one embodiment, this assay is performed utilizing
antibodies reactive with 279 protein or target molecules but which
do not interfere with binding of the 279 protein to its target
molecule. Such antibodies can be derivatized to the wells of the
plate, and unbound target or 279 protein trapped in the wells by
antibody conjugation. Methods for detecting such complexes, in
addition to those described above for the GST-immobilized
complexes, include immunodetection of complexes using antibodies
reactive with the 279 protein or target molecule, as well as
enzyme-linked assays which rely on detecting an enzymatic activity
associated with the 279 protein or target molecule.
[0302] Alternatively, cell free assays can be conducted in a liquid
phase. In such an assay, the reaction products are separated from
unreacted components, by any of a number of standard techniques,
including but not limited to: differential centrifugation (see, for
example, Rivas, G., and Minton, A. P., (1993) Trends Biochem Sci
18:284-7); chromatography (gel filtration chromatography,
ion-exchange chromatography); electrophoresis (see, e.g., Ausubel,
F. et al., eds. Current Protocols in Molecular Biology 1999, J.
Wiley: New York.); and immunoprecipitation (see, for example,
Ausubel, F. et al., eds. (1999) Current Protocols in Molecular
Biology, J. Wiley: New York). Such resins and chromatographic
techniques are known to one skilled in the art (see, e.g.,
Heegaard, N. H., (1998) J Mol Recognit 11: 141-8; Hage, D. S., and
Tweed, S. A. (1997) J Chromatogr B Biomed Sci Appl. 699:499-525).
Further, fluorescence energy transfer may also be conveniently
utilized, as described herein, to detect binding without further
purification of the complex from solution.
[0303] In a preferred embodiment, the assay includes contacting the
279 protein or biologically active portion thereof with a known
compound which binds 279 to form an assay mixture, contacting the
assay mixture with a test compound, and determining the ability of
the test compound to interact with a 279 protein, wherein
determining the ability of the test compound to interact with a 279
protein includes determining the ability of the test compound to
preferentially bind to 279 or biologically active portion thereof,
or to modulate the activity of a target molecule, as compared to
the known compound.
[0304] The target gene products of the invention can, in vivo,
interact with one or more cellular or extracellular macromolecules,
such as proteins. For the purposes of this discussion, such
cellular and extracellular macromolecules are referred to herein as
"binding partners." Compounds that disrupt such interactions can be
useful in regulating the activity of the target gene product. Such
compounds can include, but are not limited to molecules such as
antibodies, peptides, and small molecules. The preferred target
genes/products for use in this embodiment are the 279 genes herein
identified. In an alternative embodiment, the invention provides
methods for determining the ability of the test compound to
modulate the activity of a 279 protein through modulation of the
activity of a downstream effector of a 279 target molecule. For
example, the activity of the effector molecule on an appropriate
target can be determined, or the binding of the effector to an
appropriate target can be determined, as previously described.
[0305] To identify compounds that interfere with the interaction
between the target gene product and its cellular or extracellular
binding partner(s), a reaction mixture containing the target gene
product and the binding partner is prepared, under conditions and
for a time sufficient, to allow the two products to form complex.
In order to test an inhibitory agent, the reaction mixture is
provided in the presence and absence of the test compound. The test
compound can be initially included in the reaction mixture, or can
be added at a time subsequent to the addition of the target gene
and its cellular or extracellular binding partner. Control reaction
mixtures are incubated without the test compound or with a placebo.
The formation of any complexes between the target gene product and
the cellular or extracellular binding partner is then detected. The
formation of a complex in the control reaction, but not in the
reaction mixture containing the test compound, indicates that the
compound interferes with the interaction of the target gene product
and the interactive binding partner. Additionally, complex
formation within reaction mixtures containing the test compound and
normal target gene product can also be compared to complex
formation within reaction mixtures containing the test compound and
mutant target gene product. This comparison can be important in
those cases wherein it is desirable to identify compounds that
disrupt interactions of mutant but not normal target gene
products.
[0306] These assays can be conducted in a heterogeneous or
homogeneous format. Heterogeneous assays involve anchoring either
the target gene product or the binding partner onto a solid phase,
and detecting complexes anchored on the solid phase at the end of
the reaction. In homogeneous assays, the entire reaction is carried
out in a liquid phase. In either approach, the order of addition of
reactants can be varied to obtain different information about the
compounds being tested. For example, test compounds that interfere
with the interaction between the target gene products and the
binding partners, e.g., by competition, can be identified by
conducting the reaction in the presence of the test substance.
Alternatively, test compounds that disrupt preformed complexes,
e.g., compounds with higher binding constants that displace one of
the components from the complex, can be tested by adding the test
compound to the reaction mixture after complexes have been formed.
The various formats are briefly described below.
[0307] In a heterogeneous assay system, either the target gene
product or the interactive cellular or extracellular binding
partner, is anchored onto a solid surface (e.g., a microtiter
plate), while the non-anchored species is labeled, either directly
or indirectly. The anchored species can be immobilized by
non-covalent or covalent attachments. Alternatively, an immobilized
antibody specific for the species to be anchored can be used to
anchor the species to the solid surface.
[0308] In order to conduct the assay, the partner of the
immobilized species is exposed to the coated surface with or
without the test compound. After the reaction is complete,
unreacted components are removed (e.g., by washing) and any
complexes formed will remain immobilized on the solid surface.
Where the non-immobilized species is pre-labeled, the detection of
label immobilized on the surface indicates that complexes were
formed. Where the non-immobilized species is not pre-labeled, an
indirect label can be used to detect complexes anchored on the
surface; e.g., using a labeled antibody specific for the initially
non-immobilized species (the antibody, in turn, can be directly
labeled or indirectly labeled with, e.g., a labeled anti-Ig
antibody). Depending upon the order of addition of reaction
components, test compounds that inhibit complex formation or that
disrupt preformed complexes can be detected.
[0309] Alternatively, the reaction can be conducted in a liquid
phase in the presence or absence of the test compound, the reaction
products separated from unreacted components, and complexes
detected; e.g., using an immobilized antibody specific for one of
the binding components to anchor any complexes formed in solution,
and a labeled antibody specific for the other partner to detect
anchored complexes. Again, depending upon the order of addition of
reactants to the liquid phase, test compounds that inhibit complex
or that disrupt preformed complexes can be identified.
[0310] In an alternate embodiment of the invention, a homogeneous
assay can be used. For example, a preformed complex of the target
gene product and the interactive cellular or extracellular binding
partner product is prepared in that either the target gene products
or their binding partners are labeled, but the signal generated by
the label is quenched due to complex formation (see, e.g., U.S.
Pat. No. 4,109,496 that utilizes this approach for immunoassays).
The addition of a test substance that competes with and displaces
one of the species from the preformed complex will result in the
generation of a signal above background. In this way, test
substances that disrupt target gene product-binding partner
interaction can be identified.
[0311] In yet another aspect, the 279 proteins can be used as "bait
proteins" in a two-hybrid assay or three-hybrid assay (see, e.g.,
U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232;
Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al.
(1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene
8:1693-1696; and Brent WO94/10300), to identify other proteins,
which bind to or interact with 279 ("279-binding proteins" or
"279-bp") and are involved in 279 activity. Such 279-bps can be
activators or inhibitors of signals by the 279 proteins or 279
targets as, for example, downstream elements of a 279-mediated
signaling pathway.
[0312] The two-hybrid system is based on the modular nature of most
transcription factors, which consist of separable DNA-binding and
activation domains. Briefly, the assay utilizes two different DNA
constructs. In one construct, the gene that codes for a 279 protein
is fused to a gene encoding the DNA binding domain of a known
transcription factor (e.g., GAL-4). In the other construct, a DNA
sequence, from a library of DNA sequences, that encodes an
unidentified protein ("prey" or "sample") is fused to a gene that
codes for the activation domain of the known transcription factor.
(Alternatively the: 279 protein can be the fused to the activator
domain.) If the "bait" and the "prey" proteins are able to
interact, in vivo, forming a 279-dependent complex, the DNA-binding
and activation domains of the transcription factor are brought into
close proximity. This proximity allows transcription of a reporter
gene (e.g., lacZ) which is operably linked to a transcriptional
regulatory site responsive to the transcription factor. Expression
of the reporter gene can be detected and cell colonies containing
the functional transcription factor can be isolated and used to
obtain the cloned gene which encodes the protein which interacts
with the 279 protein.
[0313] In another embodiment, modulators of 279 expression are
identified. For example, a cell or cell free mixture is contacted
with a candidate compound and the expression of 279 mRNA or protein
evaluated relative to the level of expression of 279 mRNA or
protein in the absence of the candidate compound. When expression
of 279 mRNA or protein is greater in the presence of the candidate
compound than in its absence, the candidate compound is identified
as a stimulator of 279 mRNA or protein expression. Alternatively,
when expression of 279 mRNA or protein is less (statistically
significantly less) in the presence of the candidate compound than
in its absence, the candidate compound is identified as an
inhibitor of 279 mRNA or protein expression. The level of 279 mRNA
or protein expression can be determined by methods described herein
for detecting 279 mRNA or protein.
[0314] In another aspect, the invention pertains to a combination
of two or more of the assays described herein. For example, a
modulating agent can be identified using a cell-based or a cell
free assay, and the ability of the agent to modulate the activity
of a 279 protein can be confirmed in vivo, e.g., in an animal such
as an animal model for angiogenesis related disorders such as
cancer, cardiovascular disorders such as atherosclerosis, disorders
involving vascular tone, and a superovulation model as described in
the appended examples.
[0315] This invention further pertains to novel agents identified
by the above-described screening assays. Accordingly, it is within
the scope of this invention to further use an agent identified as
described herein (e.g., a 279 modulating agent, an antisense 279
nucleic acid molecule, a 279-specific antibody, or a 279-binding
partner) in an appropriate animal model to determine the efficacy,
toxicity, side effects, or mechanism of action, of treatment with
such an agent. Furthermore, novel agents identified by the
above-described screening assays can be used for treatments as
described herein.
[0316] Detection Assays
[0317] Portions or fragments of the nucleic acid sequences
identified herein can be used as polynucleotide reagents. For
example, these sequences can be used to: (i) map their respective
genes on a chromosome e.g., to locate gene regions associated with
genetic disease or to associate 279 with a disease; (ii) identify
an individual from a minute biological sample (tissue typing); and
(iii) aid in forensic identification of a biological sample. These
applications are described in the subsections below.
[0318] Chromosome Mapping
[0319] The 279 nucleotide sequences or portions thereof can be used
to map the location of the 279 genes on a chromosome. This process
is called chromosome mapping. Chromosome mapping is useful in
correlating the 279 sequences with genes associated with
disease.
[0320] Briefly, 279 genes can be mapped to chromosomes by preparing
PCR primers (preferably 15-25 bp in length) from the 279 nucleotide
sequences. These primers can then be used for PCR screening of
somatic cell hybrids containing individual human chromosomes. Only
those hybrids containing the human gene corresponding to the 279
sequences will yield an amplified fragment.
[0321] A panel of somatic cell hybrids in which each cell line
contains either a single human chromosome or a small number of
human chromosomes, and a full set of mouse chromosomes, can allow
easy mapping of individual genes to specific human chromosomes.
(D'Eustachio P. et al. (1983) Science 220:919-924).
[0322] Other mapping strategies e.g., in situ hybridization
(described in Fan, Y. et al. (1990) Proc. Natl. Acad. Sci. USA,
87:6223-27), pre-screening with labeled flow-sorted chromosomes,
and pre-selection by hybridization to chromosome specific cDNA
libraries can be used to map 279 to a chromosomal location.
[0323] Fluorescence in situ hybridization (FISH) of a DNA sequence
to a metaphase chromosomal spread can further be used to provide a
precise chromosomal location in one step. The FISH technique can be
used with a DNA sequence as short as 500 or 600 bases. However,
clones larger than 1,000 bases have a higher likelihood of binding
to a unique chromosomal location with sufficient signal intensity
for simple detection. Preferably 1,000 bases, and more preferably
2,000 bases will suffice to get good results at a reasonable amount
of time. For a review of this technique, see Verma et al., Human
Chromosomes: A Manual of Basic Techniques ((1988) Pergamon Press,
New York).
[0324] Reagents for chromosome mapping can be used individually to
mark a single chromosome or a single site on that chromosome, or
panels of reagents can be used for marking multiple sites and/or
multiple chromosomes. Reagents corresponding to noncoding regions
of the genes actually are preferred for mapping purposes. Coding
sequences are more likely to be conserved within gene families,
thus increasing the chance of cross hybridizations during
chromosomal mapping.
[0325] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. (Such data are found, for
example, in V. McKusick, Mendelian Inheritance in Man, available
on-line through Johns Hopkins University Welch Medical Library).
The relationship between a gene and a disease, mapped to the same
chromosomal region, can then be identified through linkage analysis
(co-inheritance of physically adjacent genes), described in, for
example, Egeland, J. et al. (1987) Nature, 325:783-787.
[0326] Moreover, differences in the DNA sequences between
individuals affected and unaffected with a disease associated with
the 279 gene, can be determined. If a mutation is observed in some
or all of the affected individuals but not in any unaffected
individuals, then the mutation is likely to be the causative agent
of the particular disease. Comparison of affected and unaffected
individuals generally involves first looking for structural
alterations in the chromosomes, such as deletions or translocations
that are visible from chromosome spreads or detectable using PCR
based on that DNA sequence. Ultimately, complete sequencing of
genes from several individuals can be performed to confirm the
presence of a mutation and to distinguish mutations from
polymorphisms.
[0327] Tissue Typing
[0328] 279 sequences can be used to identify individuals from
biological samples using, e.g., restriction fragment length
polymorphism (RFLP). In this technique, an individual's genomic DNA
is digested with one or more restriction enzymes, the fragments
separated, e.g., in a Southern blot, and probed to yield bands for
identification. The sequences of the present invention are useful
as additional DNA markers for RFLP (described in U.S. Pat. No.
5,272,057).
[0329] Furthermore, the sequences of the present invention can also
be used to determine the actual base-by-base DNA sequence of
selected portions of an individual's genome. Thus, the 279
nucleotide sequences described herein can be used to prepare two
PCR primers from the 5' and 3' ends of the sequences. These primers
can then be used to amplify an individual's DNA and subsequently
sequence it. Panels of corresponding DNA sequences from
individuals, prepared in this manner, can provide unique individual
identifications, as each individual will have a unique set of such
DNA sequences due to allelic differences.
[0330] Allelic variation occurs to some degree in the coding
regions of these sequences, and to a greater degree in the
noncoding regions. Each of the sequences described herein can, to
some degree, be used as a standard against which DNA from an
individual can be compared for identification purposes. Because
greater numbers of polymorphisms occur in the noncoding regions,
fewer sequences are necessary to differentiate individuals. The
noncoding sequences of SEQ ID NO: 1 can provide positive individual
identification with a panel of perhaps 10 to 1,000 primers which
each yield a noncoding amplified sequence of 100 bases. If
predicted coding sequences, such as those in SEQ ID NO: 3 are used,
a more appropriate number of primers for positive individual
identification would be 500-2,000.
[0331] If a panel of reagents from 279 nucleotide sequences
described herein is used to generate a unique identification
database for an individual, those same reagents can later be used
to identify tissue from that individual. Using the unique
identification database, positive identification of the individual,
living or dead, can be made from extremely small tissue
samples.
[0332] Use of Partial 279 Sequences in Forensic Biology
[0333] DNA-based identification techniques can also be used in
forensic biology. To make such an identification, PCR technology
can be used to amplify DNA sequences taken from very small
biological samples such as tissues, e.g., hair or skin, or body
fluids, e.g., blood, saliva, or semen found at a crime scene. The
amplified sequence can then be compared to a standard, thereby
allowing identification of the origin of the biological sample.
[0334] The sequences of the present invention can be used to
provide polynucleotide reagents, e.g., PCR primers, targeted to
specific loci in the human genome, which can enhance the
reliability of DNA-based forensic identifications by, for example,
providing another "identification marker" (i.e. another DNA
sequence that is unique to a particular individual). As mentioned
above, actual base sequence information can be used for
identification as an accurate alternative to patterns formed by
restriction enzyme generated fragments. Sequences targeted to
noncoding regions of SEQ ID NO: 1 (e.g., fragments derived from the
noncoding regions of SEQ ID NO: 1 having a length of at least 20
bases, preferably at least 30 bases) are particularly appropriate
for this use.
[0335] The 279 nucleotide sequences described herein can further be
used to provide polynucleotide reagents, e.g., labeled or labelable
probes which can be used in, for example, an in situ hybridization
technique, to identify a specific tissue. This can be very useful
in cases where a forensic pathologist is presented with a tissue of
unknown origin. Panels of such 279 probes can be used to identify
tissue by species and/or by organ type.
[0336] In a similar fashion, these reagents, e.g., 279 primers or
probes can be used to screen tissue culture for contamination (i.e.
screen for the presence of a mixture of different types of cells in
a culture).
[0337] Predictive Medicine
[0338] The present invention also pertains to the field of
predictive medicine in which diagnostic assays, prognostic assays,
and monitoring clinical trials are used for prognostic (predictive)
purposes to thereby treat an individual.
[0339] Generally, the invention provides, a method of determining
if a subject is at risk for a disorder related to a lesion in or
the misexpression of a gene which-encodes 279.
[0340] Such disorders include, e.g., a disorder associated with the
misexpression of 279 gene; e.g., a disorder of the cardiovascular
system.
[0341] The method includes one or more of the following:
[0342] detecting, in a tissue of the subject, the presence or
absence of a mutation which affects the expression of the 279 gene,
or detecting the presence or absence of a mutation in a region
which controls the expression of the gene, e.g., a mutation in the
5' control region;
[0343] detecting, in a tissue of the subject, the presence or
absence of a mutation which alters the structure of the 279
gene;
[0344] detecting, in a tissue of the subject, the misexpression of
the 279 gene, at the mRNA level, e.g., detecting a non-wild type
level of a mRNA;
[0345] detecting, in a tissue of the subject, the misexpression of
the gene, at the protein level, e.g., detecting a non-wild type
level of a 279 polypeptide.
[0346] In preferred embodiments the method includes: ascertaining
the existence of at least one of: a deletion of one or more
nucleotides from the 279 gene; an insertion of one or more
nucleotides into the gene, a point mutation, e.g., a substitution
of one or more nucleotides of the gene, a gross chromosomal
rearrangement of the gene, e.g., a translocation, inversion, or
deletion.
[0347] For example, detecting the genetic lesion can include: (i)
providing a probe/primer including an oligonucleotide containing a
region of nucleotide sequence which hybridizes to a sense or
antisense sequence from SEQ ID NO: 1, or naturally occurring
mutants thereof or 5' or 3' flanking sequences naturally associated
with the 279 gene; (ii) exposing the probe/primer to nucleic acid
of the tissue; and detecting, by hybridization, e.g., in situ
hybridization, of the probe/primer to the nucleic acid, the
presence or absence of the genetic lesion.
[0348] In preferred embodiments detecting the misexpression
includes ascertaining the existence of at least one of: an
alteration in the level of a messenger RNA transcript of the 279
gene; the presence of a non-wild type splicing pattern of a
messenger RNA transcript of the gene; or a non-wild type level of
279.
[0349] Methods of the invention can be used prenatally or to
determine if a subject's offspring will be at risk for a
disorder.
[0350] In preferred embodiments the method includes determining the
structure of a 279 gene, an abnormal structure being indicative of
risk for the disorder.
[0351] In preferred embodiments the method includes contacting a
sample from the subject with an antibody to the 279 protein or a
nucleic acid, which hybridizes specifically with the gene. These
and other embodiments are discussed below.
[0352] Diagnostic and Prognostic Assays
[0353] Diagnostic and prognostic assays of the invention include
method for assessing the expression level of 279 molecules and for
identifying variations and mutations in the sequence of 279
molecules.
[0354] Expression Monitoring and Profiling. The presence, level, or
absence of 279 protein or nucleic acid in a biological sample can
be evaluated by obtaining a biological sample from a test subject
and contacting the biological sample with a compound or an agent
capable of detecting 279 protein or nucleic acid (e.g., mRNA,
genomic DNA) that encodes 279 protein such that the presence of 279
protein or nucleic acid is detected in the biological sample. The
term "biological sample" includes tissues, cells and biological
fluids isolated from a subject, as well as tissues, cells and
fluids present within a subject. A preferred biological sample is
serum. The level of expression of the 279 gene can be measured in a
number of ways, including, but not limited to: measuring the mRNA
encoded by the 279 genes; measuring the amount of protein encoded
by the 279 genes; or measuring the activity of the protein encoded
by the 279 genes.
[0355] The level of mRNA corresponding to the 279 gene in a cell
can be determined both by in situ and by in vitro formats.
[0356] The isolated mRNA can be used in hybridization or
amplification assays that include, but are not limited to, Southern
or Northern analyses, polymerase chain reaction analyses and probe
arrays. One preferred diagnostic method for the detection of mRNA
levels involves contacting the isolated mRNA with a nucleic acid
molecule (probe) that can hybridize to the mRNA encoded by the gene
being detected. The nucleic acid probe can be, for example, a
full-length 279 nucleic acid, such as the nucleic acid of SEQ ID
NO: 1, or a portion thereof, such as an oligonucleotide of at least
7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient
to specifically hybridize under stringent conditions to 279 mRNA or
genomic DNA. The probe can be disposed on an address of an array,
e.g., an array described below. Other suitable probes for use in
the diagnostic assays are described herein.
[0357] In one format, mRNA (or cDNA) is immobilized on a surface
and contacted with the probes, for example by running the isolated
mRNA on an agarose gel and transferring the mRNA from the gel to a
membrane, such as nitrocellulose. In an alternative format, the
probes are immobilized on a surface and the mRNA (or cDNA) is
contacted with the probes, for example, in a two-dimensional gene
chip array described below. A skilled artisan can adapt known mRNA
detection methods for use in detecting the level of mRNA encoded by
the 279 genes.
[0358] The level of mRNA in a sample that is encoded by one of 279
can be evaluated with nucleic acid amplification, e.g., by rtPCR
(Mullis (1987) U.S. Pat. No. 4,683,202), ligase chain reaction
(Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self
sustained sequence replication (Guatelli et al., (1990) Proc. Natl.
Acad. Sci. USA 87:1874-1878), transcriptional amplification system
(Kwoh et al., (1989), Proc. Natl. Acad. Sci. USA 86:1173-1177),
Q-Beta Replicase (Lizardi et al., (1988) Bio/Technology 6:1197),
rolling circle replication (Lizardi et al., U.S. Pat. No.
5,854,033) or any other nucleic acid amplification method, followed
by the detection of the amplified molecules using techniques known
in the art. As used herein, amplification primers are defined as
being a pair of nucleic acid molecules that can anneal to 5' or 3'
regions of a gene (plus and minus strands, respectively, or
vice-versa) and contain a short region in between. In general,
amplification primers are from about 10 to 30 nucleotides in length
and flank a region from about 50 to 200 nucleotides in length.
Under appropriate conditions and with appropriate reagents, such
primers permit the amplification of a nucleic acid molecule
comprising the nucleotide sequence flanked by the primers.
[0359] For in situ methods, a cell or tissue sample can be
prepared/processed and immobilized on a support, typically a glass
slide, and then contacted with a probe that can hybridize to mRNA
that encodes the 279 gene being analyzed.
[0360] In another embodiment, the methods further contacting a
control sample with a compound or agent capable of detecting 279
mRNA, or genomic DNA, and comparing the presence of 279 mRNA or
genomic DNA in the control sample with the presence of 279 mRNA or
genomic DNA in the test sample. In still another embodiment, serial
analysis of gene expression, as described in U.S. Pat. No.
5,695,937, is used to detect 279 transcript levels.
[0361] A variety of methods can be used to determine the level of
protein encoded by 279. In general, these methods include
contacting an agent that selectively binds to the protein, such as
an antibody with a sample, to evaluate the level of protein in the
sample. In a preferred embodiment, the antibody bears a detectable
label. Antibodies can be polyclonal, or more preferably,
monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or
F(ab').sub.2) can be used. The term "labeled", with regard to the
probe or antibody, is intended to encompass direct labeling of the
probe or antibody by coupling (i.e., physically linking) a
detectable substance to the probe or antibody, as well as indirect
labeling of the probe or antibody by reactivity with a detectable
substance. Examples of detectable substances are provided
herein.
[0362] The detection methods can be used to detect 279 protein in a
biological sample in vitro as well as in vivo. In vitro techniques
for detection of 279 protein include enzyme linked immunosorbent
assays (ELISAs), immunoprecipitations, immunofluorescence, enzyme
immunoassay (EIA), radioimmunoassay (RIA), and Western blot
analysis. In vivo techniques for detection of 279 protein include
introducing into a subject a labeled anti-279 antibody. For
example, the antibody can be labeled with a radioactive marker
whose presence and location in a subject can be detected by
standard imaging techniques. In another embodiment, the sample is
labeled, e.g., biotinylated and then contacted to the antibody,
e.g., an anti-279 antibody positioned on an antibody array (as
described below). The sample can be detected, e.g., with avidin
coupled to a fluorescent label.
[0363] In another embodiment, the methods further include
contacting the control sample with a compound or agent capable of
detecting 279 protein, and comparing the presence of 279 protein in
the control sample with the presence of 279 protein in the test
sample.
[0364] The invention also includes kits for detecting the presence
of 279 in a biological sample. For example, the kit can include a
compound or agent capable of detecting 279 protein or mRNA in a
biological sample; and a standard. The compound or agent can be
packaged in a suitable container. The kit can further comprise
instructions for using the kit to detect 279 protein or nucleic
acid.
[0365] For antibody-based kits, the kit can include: (1) a first
antibody (e.g., attached to a solid support) which binds to a
polypeptide corresponding to a marker of the invention; and,
optionally, (2) a second, different antibody which binds to either
the polypeptide or the first antibody and is conjugated to a
detectable agent.
[0366] For oligonucleotide-based kits, the kit can include: (1) an
oligonucleotide, e.g., a detectably labeled oligonucleotide, which
hybridizes to a nucleic acid sequence encoding a polypeptide
corresponding to a marker of the invention or (2) a pair of primers
useful for amplifying a nucleic acid molecule corresponding to a
marker of the invention. The kit can also includes a buffering
agent, a preservative, or a protein stabilizing agent. The kit can
also includes components necessary for detecting the detectable
agent (e.g., an enzyme or a substrate). The kit can also contain a
control sample or a series of control samples which can be assayed
and compared to the test sample contained. Each component of the
kit can be enclosed within an individual container and all of the
various containers can be within a single package, along with
instructions for interpreting the results of the assays performed
using the kit.
[0367] The diagnostic methods described herein can identify
subjects having, or at risk of developing, a disease or disorder
associated with misexpressed or aberrant or unwanted 279 expression
or activity. As used herein, the term "unwanted" includes an
unwanted phenomenon involved in a biological response such as
angiogenesis related disorders including cancer, cardiovascular
disorders including atherosclerosis, disorders involving vascular
tone, or deregulated cell proliferation.
[0368] In one embodiment, a disease or disorder associated with
aberrant or unwanted 279 expression or activity is identified. A
test sample is obtained from a subject and 279 protein or nucleic
acid (e.g., mRNA or genomic DNA) is evaluated, wherein the level,
e.g., the presence or absence, of 279 protein or nucleic acid is
diagnostic for a subject having or at risk of developing a disease
or disorder associated with aberrant or unwanted 279 expression or
activity. As used herein, a "test sample" refers to a biological
sample obtained from a subject of interest, including a biological
fluid (e.g., serum), cell sample, or tissue.
[0369] The prognostic assays described herein can be used to
determine whether a subject can be administered an agent (e.g., an
agonist, antagonist, peptidomimetic, protein, peptide, nucleic
acid, small molecule, or other drug candidate) to treat a disease
or disorder associated with aberrant or unwanted 279 expression or
activity. For example, such methods can be used to determine
whether a subject can be effectively treated with an agent for an
angiogenesis related disorders such as cancer, a cardiovascular
disorder such as atherosclerosis, or a disorder involving vascular
tone.
[0370] In another aspect, the invention features a computer medium
having a plurality of digitally encoded data records. Each data
record includes a value representing the level of expression of 279
in a sample, and a descriptor of the sample. The descriptor of the
sample can be an identifier of the sample, a subject from which the
sample was derived (e.g., a patient), a diagnosis, or a treatment
(e.g., a preferred treatment). In a preferred embodiment, the data
record further includes values representing the level of expression
of genes other than 279 (e.g., other genes associated with a
279-disorder, or other genes on an array). The data record can be
structured as a table, e.g., a table that is part of a database
such as a relational database (e.g., a SQL database of the Oracle
or Sybase database environments).
[0371] Also featured is a method of evaluating a sample. The method
includes providing a sample, e.g., from the subject, and
determining a gene expression profile of the sample, wherein the
profile includes a value representing the level of 279 expression.
The method can further include comparing the value or the profile
(i.e., multiple values) to a reference value or reference profile.
The gene expression profile of the sample can be obtained by any of
the methods described herein (e.g., by providing a nucleic acid
from the sample and contacting the nucleic acid to an array). The
method can be used to diagnose a cardiovascular disorder in a
subject wherein an increase in 279 expression is an indication that
the subject has or is disposed to having atherosclerosis. The
method can be used to monitor a treatment for angiogenesis
disorders such as cancer, cardiovascular disorders such as
atherosclerosis and disorders involving vascular tone in a subject.
For example, the gene expression profile can be determined for a
sample from a subject undergoing treatment. The profile can be
compared to a reference profile or to a profile obtained from the
subject prior to treatment or prior to onset of the disorder (see,
e.g., Golub et al. (1999) Science 286:531).
[0372] In yet another aspect, the invention features a method of
evaluating a test compound (see also, "Screening Assays", above).
The method includes providing a cell and a test compound;
contacting the test compound to the cell; obtaining a subject
expression profile for the contacted cell; and comparing the
subject expression profile to one or more reference profiles. The
profiles include a value representing the level of 279 expression.
In a preferred embodiment, the subject expression profile is
compared to a target profile, e.g., a profile for a normal cell or
for desired condition of a cell. The test compound is evaluated
favorably if the subject expression profile is more similar to the
target profile than an expression profile obtained from an
uncontacted cell.
[0373] In another aspect, the invention features, a method of
evaluating a subject. The method includes: a) obtaining a sample
from a subject, e.g., from a caregiver, e.g., a caregiver who
obtains the sample from the subject; b) determining a subject
expression profile for the sample. Optionally, the method further
includes either or both of steps: c) comparing the subject
expression profile to one or more reference expression profiles;
and d) selecting the reference profile most similar to the subject
reference profile. The subject expression profile and the reference
profiles include a value representing the level of 279 expression.
A variety of routine statistical measures can be used to compare
two reference profiles. One possible metric is the length of the
distance vector that is the difference between the two profiles.
Each of the subject and reference profile is represented as a
multi-dimensional vector, wherein each dimension is a value in the
profile.
[0374] The method can further include transmitting a result to a
caregiver. The result can be the subject expression profile, a
result of a comparison of the subject expression profile with
another profile, a most similar reference profile, or a descriptor
of any of the aforementioned. The result can be transmitted across
a computer network, e.g., the result can be in the form of a
computer transmission, e.g., a computer data signal embedded in a
carrier wave.
[0375] Also featured is a computer medium having executable code
for effecting the following steps: receive a subject expression
profile; access a database of reference expression profiles; and
either i) select a matching reference profile most similar to the
subject expression profile or ii) determine at least one comparison
score for the similarity of the subject expression profile to at
least one reference profile. The subject expression profile, and
the reference expression profiles each include a value representing
the level of 279 expression.
[0376] Arrays and Uses Thereof
[0377] In another aspect, the invention features an array that
includes a substrate having a plurality of addresses. At least one
address of the plurality includes a capture probe that binds
specifically to a 279 molecule (e.g., a 279 nucleic acid or a 279
polypeptide). The array can have a density of at least than 10, 50,
100, 200, 500, 1,000, 2,000, or 10,000 or more addresses/cm.sup.2,
and ranges between. In a preferred embodiment, the plurality of
addresses includes at least 10, 100, 500, 1,000, 5,000, 10,000,
50,000 addresses. In a preferred embodiment, the plurality of
addresses includes equal to or less than 10, 100, 500, 1,000,
5,000, 10,000, or 50,000 addresses. The substrate can be a
two-dimensional substrate such as a glass slide, a wafer (e.g.,
silica or plastic), a mass spectroscopy plate, or a
three-dimensional substrate such as a gel pad. Addresses in
addition to address of the plurality can be disposed on the
array.
[0378] In a preferred embodiment, at least one address of the
plurality includes a nucleic acid capture probe that hybridizes
specifically to a 279 nucleic acid, e.g., the sense or anti-sense
strand. In one preferred embodiment, a subset of addresses of the
plurality of addresses has a nucleic acid capture probe for 279.
Each address of the subset can include a capture probe that
hybridizes to a different region of a 279 nucleic acid. In another
preferred embodiment, addresses of the subset include a capture
probe for a 279 nucleic acid. Each address of the subset is unique,
overlapping, and complementary to a different variant of 279 (e.g.,
an allelic variant, or all possible hypothetical variants). The
array can be used to sequence 279 by hybridization (see, e.g., U.S.
Pat. No. 5,695,940).
[0379] An array can be generated by various methods, e.g., by
photolithographic methods (see, e.g., U.S. Pat. Nos. 5,143,854;
5,510,270; and 5,527,681), mechanical methods (e.g., directed-flow
methods as described in U.S. Pat. No. 5,384,261), pin-based methods
(e.g., as described in U.S. Pat. No. 5,288,514), and bead-based
techniques (e.g., as described in PCT US/93/04145).
[0380] In another preferred embodiment, at least one address of the
plurality includes a polypeptide capture probe that binds
specifically to a 279 polypeptide or fragment thereof. The
polypeptide can be a naturally-occurring interaction partner of 279
polypeptide. Preferably, the polypeptide is an antibody, e.g., an
antibody described herein (see "Anti-279 Antibodies," above), such
as a monoclonal antibody or a single-chain antibody.
[0381] In another aspect, the invention features a method of
analyzing the expression of 279. The method includes providing an
array as described above; contacting the array with a sample and
detecting binding of a 279-molecule (e.g., nucleic acid or
polypeptide) to the array. In a preferred embodiment, the array is
a nucleic acid array. Optionally the method further includes
amplifying nucleic acid from the sample prior or during contact
with the array.
[0382] In another embodiment, the array can be used to assay gene
expression in a tissue to ascertain tissue specificity of genes in
the array, particularly the expression of 279. If a sufficient
number of diverse samples is analyzed, clustering (e.g.,
hierarchical clustering, k-means clustering, Bayesian clustering
and the like) can be used to identify other genes which are
co-regulated with 279. For example, the array can be used for the
quantitation of the expression of multiple genes. Thus, not only
tissue specificity, but also the level of expression of a battery
of genes in the tissue is ascertained. Quantitative data can be
used to group (e.g., cluster) genes on the basis of their tissue
expression per se and level of expression in that tissue.
[0383] For example, array analysis of gene expression can be used
to assess the effect of cell-cell interactions on 279 expression. A
first tissue can be perturbed and nucleic acid from a second tissue
that interacts with the first tissue can be analyzed. In this
context, the effect of one cell type on another cell type in
response to a biological stimulus can be determined, e.g., to
monitor the effect of cell-cell interaction at the level of gene
expression.
[0384] In another embodiment, cells are contacted with a
therapeutic agent. The expression profile of the cells is
determined using the array, and the expression profile is compared
to the profile of like cells not contacted with the agent. For
example, the assay can be used to determine or analyze the
molecular basis of an undesirable effect of the therapeutic agent.
If an agent is administered therapeutically to treat one cell type
but has an undesirable effect on another cell type, the invention
provides an assay to determine the molecular basis of the
undesirable effect and thus provides the opportunity to
co-administer a counteracting agent or otherwise treat the
undesired effect. Similarly, even within a single cell type,
undesirable biological effects can be determined at the molecular
level. Thus, the effects of an agent on expression of other than
the target gene can be ascertained and counteracted.
[0385] In another embodiment, the array can be used to monitor
expression of one or more genes in the array with respect to time.
For example, samples obtained from different time points can be
probed with the array. Such analysis can identify and/or
characterize the development of a 279-associated disease or
disorder; and processes, such as a cellular transformation
associated with a 279-associated disease or disorder. The method
can also evaluate the treatment and/or progression of a
279-associated disease or disorder
[0386] The array is also useful for ascertaining differential
expression patterns of one or more genes in normal and abnormal
cells. This provides a battery of genes (e.g., including 279) that
could serve as a molecular target for diagnosis or therapeutic
intervention.
[0387] In another aspect, the invention features an array having a
plurality of addresses. Each address of the plurality includes a
unique polypeptide. At least one address of the plurality has
disposed thereon a 279 polypeptide or fragment thereof. Methods of
producing polypeptide arrays are described in the art, e.g., in De
Wildt et al. (2000). Nature Biotech. 18, 989-994; Lueking et al.
(1999). Anal. Biochem. 270, 103-1 11; Ge, H. (2000). Nucleic Acids
Res. 28, e3, I-VII; MacBeath, G., and Schreiber, S.L. (2000).
Science 289, 1760-1763; and WO 99/51773A1. In a preferred
embodiment, each addresses of the plurality has disposed thereon a
polypeptide at least 60, 70, 80, 85, 90, 95 or 99% identical to a
279 polypeptide or fragment thereof. For example, multiple variants
of a 279 polypeptide (e.g., encoded by allelic variants,
site-directed mutants, random mutants, or combinatorial mutants)
can be disposed at individual addresses of the plurality. Addresses
in addition to the address of the plurality can be disposed on the
array.
[0388] The polypeptide array can be used to detect a 279 binding
compound, e.g., an antibody in a sample from a subject with
specificity for a 279 polypeptide or the presence of a 279-binding
protein or ligand.
[0389] The array is also useful for ascertaining the effect of the
expression of a gene on the expression of other genes in the same
cell or in different cells (e.g., ascertaining the effect of 279
expression on the expression of other genes). This provides, for
example, for a selection of alternate molecular targets for
therapeutic intervention if the ultimate or downstream target
cannot be regulated.
[0390] In another aspect, the invention features a method of
analyzing a plurality of probes. The method is useful, e.g., for
analyzing gene expression. The method includes: providing a two
dimensional array having a plurality of addresses, each address of
the plurality being positionally distinguishable from each other
address of the plurality having a unique capture probe, e.g.,
wherein the capture probes are from a cell or subject which express
279 or from a cell or subject in which a 279 mediated response has
been elicited, e.g., by contact of the cell with 279 nucleic acid
or protein, or administration to the cell or subject 279 nucleic
acid or protein; providing a two dimensional array having a
plurality of addresses, each address of the plurality being
positionally distinguishable from each other address of the
plurality, and each address of the plurality having a unique
capture probe, e.g., wherein the capture probes are from a cell or
subject which does not express 279 (or does not express as highly
as in the case of the 279 positive plurality of capture probes) or
from a cell or subject which in which a 279 mediated response has
not been elicited (or has been elicited to a lesser extent than in
the first sample); contacting the array with one or more inquiry
probes (which is preferably other than a 279 nucleic acid,
polypeptide, or antibody), and thereby evaluating the plurality of
capture probes. Binding, e.g., in the case of a nucleic acid,
hybridization with a capture probe at an address of the plurality,
is detected, e.g., by signal generated from a label attached to the
nucleic acid, polypeptide, or antibody.
[0391] In another aspect, the invention features a method of
analyzing a plurality of probes or a sample. The method is useful,
e.g., for analyzing gene expression. The method includes: providing
a two dimensional array having a plurality of addresses, each
address of the plurality being positionally distinguishable from
each other address of the plurality having a unique capture probe,
contacting the array with a first sample from a cell or subject
which express or mis-express 279 or from a cell or subject in which
a 279-mediated response has been elicited, e.g., by contact of the
cell with 279 nucleic acid or protein, or administration to the
cell or subject 279 nucleic acid or protein; providing a two
dimensional array having a plurality of addresses, each address of
the plurality being positionally distinguishable from each other
address of the plurality, and each address of the plurality having
a unique capture probe, and contacting the array with a second
sample from a cell or subject which does not express 279 (or does
not express as highly as in the case of the 279 positive plurality
of capture probes) or from a cell or subject which in which a 279
mediated response has not been elicited (or has been elicited to a
lesser extent than in the first sample); and comparing the binding
of the first sample with the binding of the second sample. Binding,
e.g., in the case of a nucleic acid, hybridization with a capture
probe at an address of the plurality, is detected, e.g., by signal
generated from a label attached to the nucleic acid, polypeptide,
or antibody. A The same array can be used for both samples or
different arrays can be used. If different arrays are used the
plurality of addresses with capture probes should be present on
both arrays.
[0392] In another aspect, the invention features a method of
analyzing 279, e.g., analyzing structure, function, or relatedness
to other nucleic acid or amino acid sequences. The method includes:
providing a 279 nucleic acid or amino acid sequence; comparing the
279 sequence with one or more preferably a plurality of sequences
from a collection of sequences, e.g., a nucleic acid or protein
sequence database; to thereby analyze 279.
[0393] Detection of Variations or Mutations
[0394] The methods of the invention can also be used to detect
genetic alterations in a 279 gene, thereby determining if a subject
with the altered gene is at risk for a disorder characterized by
misregulation in 279 protein activity or nucleic acid expression,
such as an angiogenesis related disorders such as cancer,
cardiovascular disorders such as atherosclerosis and disorders
involving vascular tone disorder. In preferred embodiments, the
methods include detecting, in a sample from the subject, the
presence or absence of a genetic alteration characterized by at
least one of an alteration affecting the integrity of a gene
encoding a 279-protein, or the mis-expression of the 279 gene. For
example, such genetic alterations can be detected by ascertaining
the existence of at least one of 1) a deletion of one or more
nucleotides from a 279 gene; 2) an addition of one or more
nucleotides to a 279 gene; 3) a substitution of one or more
nucleotides of a 279 gene, 4) a chromosomal rearrangement of a 279
gene; 5) an alteration in the level of a messenger RNA transcript
of a 279 gene, 6) aberrant modification of a 279 gene, such as of
the methylation pattern of the genomic DNA, 7) the presence of a
non-wild type splicing pattern of a messenger RNA transcript of a
279 gene, 8) a non-wild type level of a 279-protein, 9) allelic
loss of a 279 gene, and 10) inappropriate post-translational
modification of a 279-protein.
[0395] An alteration can be detected without a probe/primer in a
polymerase chain reaction, such as anchor PCR or RACE PCR, or,
alternatively, in a ligation chain reaction (LCR), the latter of
which can be particularly useful for detecting point mutations in
the 279-gene. This method can include the steps of collecting a
sample of cells from a subject, isolating nucleic acid (e.g.,
genomic, mRNA or both) from the sample, contacting the nucleic acid
sample with one or more primers which specifically hybridize to a
279 gene under conditions such that hybridization and amplification
of the 279-gene (if present) occurs, and detecting the presence or
absence of an amplification product, or detecting the size of the
amplification product and comparing the length to a control sample.
It is anticipated that PCR and/or LCR may be desirable to use as a
preliminary amplification step in conjunction with any of the
techniques used for detecting mutations described herein.
Alternatively, other amplification methods described herein or
known in the art can be used.
[0396] In another embodiment, mutations in a 279 gene from a sample
cell can be identified by detecting alterations in restriction
enzyme cleavage patterns. For example, sample and control DNA is
isolated, amplified (optionally), digested with one or more
restriction endonucleases, and fragment length sizes are
determined, e.g., by gel electrophoresis and compared. Differences
in fragment length sizes between sample and control DNA indicates
mutations in the sample DNA. Moreover, the use of sequence specific
ribozymes (see, for example, U.S. Pat. No. 5,498,531) can be used
to score for the presence of specific mutations by development or
loss of a ribozyme cleavage site.
[0397] In other embodiments, genetic mutations in 279 can be
identified by hybridizing a sample and control nucleic acids, e.g.,
DNA or RNA, two-dimensional arrays, e.g., chip based arrays. Such
arrays include a plurality of addresses, each of which is
positionally distinguishable from the other. A different probe is
located at each address of the plurality. A probe can be
complementary to a region of a 279 nucleic acid or a putative
variant (e.g., allelic variant) thereof. A probe can have one or
more mismatches to a region of a 279 nucleic acid (e.g., a
destabilizing mismatch). The arrays can have a high density of
addresses, e.g., can contain hundreds or thousands of
oligonucleotides probes (Cronin, M. T. et al. (1996) Human Mutation
7: 244-255; Kozal, M. J. et al. (1996) Nature Medicine 2: 753-759).
For example, genetic mutations in 279 can be identified in
two-dimensional arrays containing light-generated DNA probes as
described in Cronin, M. T. et al. supra. Briefly, a first
hybridization array of probes can be used to scan through long
stretches of DNA in a sample and control to identify base changes
between the sequences by making linear arrays of sequential
overlapping probes. This step allows the identification of point
mutations. This step is followed by a second hybridization array
that allows the characterization of specific mutations by using
smaller, specialized probe arrays complementary to all variants or
mutations detected. Each mutation array is composed of parallel
probe sets, one complementary to the wild-type gene and the other
complementary to the mutant gene.
[0398] In yet another embodiment, any of a variety of sequencing
reactions known in the art can be used to directly sequence the 279
gene and detect mutations by comparing the sequence of the sample
279 with the corresponding wild-type (control) sequence. Automated
sequencing procedures can be utilized when performing the
diagnostic assays ((1995) Biotechniques 19:448), including
sequencing by mass spectrometry.
[0399] Other methods for detecting mutations in the 279 gene
include methods in which protection from cleavage agents is used to
detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers
et al. (1985) Science 230:1242; Cotton et al. (1988) Proc. Natl
Acad Sci USA 85:4397; Saleeba et al. (1992) Methods Enzymol.
217:286-295).
[0400] In still another embodiment, the mismatch cleavage reaction
employs one or more proteins that recognize mismatched base pairs
in double-stranded DNA (so called "DNA mismatch repair" enzymes) in
defined systems for detecting and mapping point mutations in 279
cDNAs obtained from samples of cells. For example, the mutY enzyme
of E. coli cleaves A at G/A mismatches and the thymidine DNA
glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al.
(1994) Carcinogenesis 15:1657-1662; U.S. Pat. No. 5,459,039).
[0401] In other embodiments, alterations in electrophoretic
mobility will be used to identify mutations in 279 genes. For
example, single strand conformation polymorphism (SSCP) may be used
to detect differences in electrophoretic mobility between mutant
and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad.
Sci USA: 86:2766, see also Cotton (1993) Mutat. Res. 285:125-144;
and Hayashi (1992) Genet. Anal. Tech. Appl. 9:73-79).
Single-stranded DNA fragments of sample and control 279 nucleic
acids will be denatured and allowed to renature. The secondary
structure of single-stranded nucleic acids varies according to
sequence, the resulting alteration in electrophoretic mobility
enables the detection of even a single base change. The DNA
fragments may be labeled or detected with labeled probes. The
sensitivity of the assay may be enhanced by using RNA (rather than
DNA), in which the secondary structure is more sensitive to a
change in sequence. In a preferred embodiment, the subject method
utilizes heteroduplex analysis to separate double stranded
heteroduplex molecules on the basis of changes in electrophoretic
mobility (Keen et al. (1991) Trends Genet 7:5).
[0402] In yet another embodiment, the movement of mutant or
wild-type fragments in polyacrylamide gels containing a gradient of
denaturant is assayed using denaturing gradient gel electrophoresis
(DGGE) (Myers et al. (1985) Nature 313:495). When DGGE is used as
the method of analysis, DNA will be modified to insure that it does
not completely denature, for example by adding a GC clamp of
approximately 40 bp of high-melting GC-rich DNA by PCR. In a
further embodiment, a temperature gradient is used in place of a
denaturing gradient to identify differences in the mobility of
control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem
265:12753).
[0403] Examples of other techniques for detecting point mutations
include, but are not limited to, selective oligonucleotide
hybridization, selective amplification, or selective primer
extension (Saiki et al. (1986) Nature 324:163); Saiki et al. (1989)
Proc. Natl Acad. Sci USA 86:6230). A further method of detecting
point mutations is the chemical ligation of oligonucleotides as
described in Xu et al. ((2001) Nature Biotechnol. 19:148). Adjacent
oligonucleotides, one of which selectively anneals to the query
site, are ligated together if the nucleotide at the query site of
the sample nucleic acid is complementary to the query
oligonucleotide; ligation can be monitored, e.g., by fluorescent
dyes coupled to the oligonucleotides.
[0404] Alternatively, allele specific amplification technology that
depends on selective PCR amplification may be used in conjunction
with the instant invention. Oligonucleotides used as primers for
specific amplification may carry the mutation of interest in the
center of the molecule (so that amplification depends on
differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res.
17:2437-2448) or at the extreme 3' end of one primer where, under
appropriate conditions, mismatch can prevent, or reduce polymerase
extension (Prossner (1993) Tibtech 11:238). In addition it may be
desirable to introduce a novel restriction site in the region of
the mutation to create cleavage-based detection (Gasparini et al.
(1992) Mol. Cell Probes 6: 1). It is anticipated that in certain
embodiments amplification may also be performed using Taq ligase
for amplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189).
In such cases, ligation will occur only if there is a perfect match
at the 3' end of the 5' sequence making it possible to detect the
presence of a known mutation at a specific site by looking for the
presence or absence of amplification.
[0405] In another aspect, the invention features a set of
oligonucleotides. The set includes a plurality of oligonucleotides,
each of which is at least partially complementary (e.g., at least
50%, 60%, 70%, 80%, 90%, 92%, 95%, 97%, 98%, or 99% complementary)
to a 279 nucleic acid.
[0406] In a preferred embodiment the set includes a first and a
second oligonucleotide. The first and second oligonucleotide can
hybridize to the same or to different locations of SEQ ID NO: 1 or
the complement of SEQ ID NO: 1. Different locations can be
different but overlapping, or non-overlapping on the same strand.
The first and second oligonucleotide can hybridize to sites on the
same or on different strands.
[0407] The set can be useful, e.g., for identifying SNP's, or
identifying specific alleles of 279. In a preferred embodiment,
each oligonucleotide of the set has a different nucleotide at an
interrogation position. In one embodiment, the set includes two
oligonucleotides, each complementary to a different allele at a
locus, e.g., a biallelic or polymorphic locus.
[0408] In another embodiment, the set includes four
oligonucleotides, each having a different nucleotide (e.g.,
adenine, guanine, cytosine, or thymidine) at the interrogation
position. The interrogation position can be a SNP or the site of a
mutation. In another preferred embodiment, the oligonucleotides of
the plurality are identical in sequence to one another (except for
differences in length). The oligonucleotides can be provided with
differential labels, such that an oligonucleotide that hybridizes
to one allele provides a signal that is distinguishable from an
oligonucleotide that hybridizes to a second allele. In still
another embodiment, at least one of the oligonucleotides of the set
has a nucleotide change at a position in addition to a query
position, e.g., a destabilizing mutation to decrease the T.sub.m of
the oligonucleotide. In another embodiment, at least one
oligonucleotide of the set has a non-natural nucleotide, e.g.,
inosine. In a preferred embodiment, the oligonucleotides are
attached to a solid support, e.g., to different addresses of an
array or to different beads or nanoparticles.
[0409] In a preferred embodiment the set of oligo nucleotides can
be used to specifically amplify, e.g., by PCR, or detect, a 279
nucleic acid.
[0410] The methods described herein may be performed, for example,
by utilizing pre-packaged diagnostic kits comprising at least one
probe nucleic acid or antibody reagent described herein, which may
be conveniently used, e.g., in clinical settings to diagnose
patients exhibiting symptoms or family history of a disease or
illness involving a 279 gene.
[0411] Use of 279 Molecules as Surrogate Markers
[0412] The 279 molecules of the invention are also useful as
markers of disorders or disease states, as markers for precursors
of disease states, as markers for predisposition of disease states,
as markers of drug activity, or as markers of the pharmacogenomic
profile of a subject. Using the methods described herein, the
presence, absence and/or quantity of the 279 molecules of the
invention may be detected, and may be correlated with one or more
biological states in vivo. For example, the 279 molecules of the
invention may serve as surrogate markers for one or more disorders
or disease states or for conditions leading up to disease states.
As used herein, a "surrogate marker" is an objective biochemical
marker which correlates with the absence or presence of a disease
or disorder, or with the progression of a disease or disorder
(e.g., with the presence or absence of a tumor). The presence or
quantity of such markers is independent of the disease. Therefore,
these markers may serve to indicate whether a particular course of
treatment is effective in lessening a disease state or disorder.
Surrogate markers are of particular use when the presence or extent
of a disease state or disorder is difficult to assess through
standard methodologies (e.g., early stage tumors), or when an
assessment of disease progression is desired before a potentially
dangerous clinical endpoint is reached (e.g., an assessment of
cardiovascular disease may be made using cholesterol levels as a
surrogate marker, and an analysis of HIV infection may be made
using HIV RNA levels as a surrogate marker, well in advance of the
undesirable clinical outcomes of myocardial infarction or
fully-developed AIDS). Examples of the use of surrogate markers in
the art include: Koomen et al. (2000) J. Mass. Spectrom. 35:
258-264; and James (1994) AIDS Treatment News Archive 209.
[0413] The 279 molecules of the invention are also useful as
pharmacodynamic markers. As used herein, a "pharmacodynamic marker"
is an objective biochemical marker which correlates specifically
with drug effects. The presence or quantity of a pharmacodynamic
marker is not related to the disease state or disorder for which
the drug is being administered; therefore, the presence or quantity
of the marker is indicative of the presence or activity of the drug
in a subject. For example, a pharmacodynamic marker may be
indicative of the concentration of the drug in a biological tissue,
in that the marker is either expressed or transcribed or not
expressed or transcribed in that tissue in relationship to the
level of the drug. In this fashion, the distribution or uptake of
the drug may be monitored by the pharmacodynamic marker. Similarly,
the presence or quantity of the pharmacodynamic marker may be
related to the presence or quantity of the metabolic product of a
drug, such that the presence or quantity of the marker is
indicative of the relative breakdown rate of the drug in vivo.
Pharmacodynamic markers are of particular use in increasing the
sensitivity of detection of drug effects, particularly when the
drug is administered in low doses. Since even a small amount of a
drug may be sufficient to activate multiple rounds of marker (e.g.,
a 279 marker) transcription or expression, the amplified marker may
be in a quantity which is more readily detectable than the drug
itself. Also, the marker may be more easily detected due to the
nature of the marker itself; for example, using the methods
described herein, anti-279 antibodies may be employed in an
immune-based detection system for a 279 protein marker, or
279-specific radiolabeled probes may be used to detect a 279 mRNA
marker. Furthermore, the use of a pharmacodynamic marker may offer
mechanism-based prediction of risk due to drug treatment beyond the
range of possible direct observations. Examples of the use of
pharmacodynamic markers in the art include: Matsuda et al. U.S.
Pat. No. 6,033,862; Hattis et al. (1991) Env. Health Perspect. 90:
229-238; Schentag (1999) Am. J. Health-Syst. Pharm. 56 Suppl. 3:
S21-S24; and Nicolau (1999) Am, J. Health-Syst. Pharm. 56 Suppl. 3:
S 16-S20.
[0414] The 279 molecules of the invention are also useful as
pharmacogenomic markers. As used herein, a "pharmacogenomic marker"
is an objective biochemical marker which correlates with a specific
clinical drug response or susceptibility in a subject (see, e.g.,
McLeod et al. (1999) Eur. J. Cancer 35:1650-1652). The presence or
quantity of the pharmacogenomic marker is related to the predicted
response of the subject to a specific drug or class of drugs prior
to administration of the drug. By assessing the presence or
quantity of one or more pharmacogenomic markers in a subject, a
drug therapy which is most appropriate for the subject, or which is
predicted to have a greater degree of success, may be selected. For
example, based on the presence or quantity of RNA, or protein
(e.g., 279 protein or RNA) for specific tumor markers in a subject,
a drug or course of treatment may be selected that is optimized for
the treatment of the specific tumor likely to be present in the
subject. Similarly, the presence or absence of a specific sequence
mutation in 279 DNA may correlate 279 drug response. The use of
pharmacogenomic markers therefore permits the application of the
most appropriate treatment for each subject without having to
administer the therapy.
[0415] Pharmaceutical Compositions
[0416] The nucleic acid and polypeptides, fragments thereof, as
well as anti-279 antibodies (also referred to herein as "active
compounds") of the invention can be incorporated into
pharmaceutical compositions. Such compositions typically include
the nucleic acid molecule, protein, or antibody and a
pharmaceutically acceptable carrier. As used herein the language
"pharmaceutically acceptable carrier" includes solvents, dispersion
media, coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like, compatible with
pharmaceutical administration. Supplementary active compounds can
also be incorporated into the compositions.
[0417] A pharmaceutical composition is formulated to be compatible
with its intended route of administration. Examples of routes of
administration include parenteral, e.g., intravenous, intradermal,
subcutaneous, oral (e.g., inhalation), transdermal (topical),
transmucosal, and rectal administration. Solutions or suspensions
used for parenteral, intradermal, or subcutaneous application can
include the following components: a sterile diluent such as water
for injection, saline solution, fixed oils, polyethylene glycols,
glycerine, propylene glycol or other synthetic solvents;
antibacterial agents such as benzyl alcohol or methyl parabens;
antioxidants such as ascorbic acid or sodium bisulfite; chelating
agents such as ethylenediaminetetraacetic acid; buffers such as
acetates, citrates or phosphates and agents for the adjustment of
tonicity such as sodium chloride or dextrose. pH can be adjusted
with acids or bases, such as hydrochloric acid or sodium hydroxide.
The parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
[0418] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringability exists. It should be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyetheylene glycol, and the like), and
suitable mixtures thereof. The proper fluidity can be maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0419] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle which contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, the preferred methods of preparation
are vacuum drying and freeze-drying which yields a powder of the
active ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof.
[0420] Oral compositions generally include an inert diluent or an
edible carrier. For the purpose of oral therapeutic administration,
the active compound can be incorporated with excipients and used in
the form of tablets, troches, or capsules, e.g., gelatin capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash. Pharmaceutically compatible binding agents,
and/or adjuvant materials can be included as part of the
composition. The tablets, pills, capsules, troches and the like can
contain any of the following ingredients, or compounds of a similar
nature: a binder such as microcrystalline cellulose, gum tragacanth
or gelatin; an excipient such as starch or lactose, a
disintegrating agent such as alginic acid, Primogel, or corn
starch; a lubricant such as magnesium stearate or Sterotes; a
glidant such as colloidal silicon dioxide; a sweetening agent such
as sucrose or saccharin; or a flavoring agent such as peppermint,
methyl salicylate, or orange flavoring.
[0421] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser which contains a suitable propellant, e.g., a gas such
as carbon dioxide, or a nebulizer.
[0422] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0423] The compounds can also be prepared in the form of
suppositories (e.g., with conventional suppository bases such as
cocoa butter and other glycerides) or retention enemas for rectal
delivery.
[0424] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0425] It is advantageous to formulate oral or parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the subject
to be treated; each unit containing a predetermined quantity of
active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier.
[0426] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD50 (the dose
lethal to 50% of the population) and the ED50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD50/ED50. Compounds which exhibit
high therapeutic indices are preferred. While compounds that
exhibit toxic side effects may be used, care should be taken to
design a delivery system that targets such compounds to the site of
affected tissue in order to minimize potential damage to uninfected
cells and, thereby, reduce side effects.
[0427] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED50 with little or
no toxicity. The dosage may vary within this range depending upon
the dosage form employed and the route of administration utilized.
For any compound used in the method of the invention, the
therapeutically effective dose can be estimated initially from cell
culture assays. A dose may be formulated in animal models to
achieve a circulating plasma concentration range that includes the
IC50 (i.e., the concentration of the test compound which achieves a
half-maximal inhibition of symptoms) as determined in cell culture.
Such information can be used to more accurately determine useful
doses in humans. Levels in plasma may be measured, for example, by
high performance liquid chromatography.
[0428] As defined herein, a therapeutically effective amount of
protein or polypeptide (i.e., an effective dosage) ranges from
about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25
mg/kg body weight, more preferably about 0.1 to 20 mg/kg body
weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg,
3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The
protein or polypeptide can be administered one time per week for
between about 1 to 10 weeks, preferably between 2 to 8 weeks, more
preferably between about 3 to 7 weeks, and even more preferably for
about 4, 5, or 6 weeks. The skilled artisan will appreciate that
certain factors may influence the dosage and timing required to
effectively treat a subject, including but not limited to the
severity of the disease or disorder, previous treatments, the
general health and/or age of the subject, and other diseases
present. Moreover, treatment of a subject with a therapeutically
effective amount of a protein, polypeptide, or antibody can include
a single treatment or, preferably, can include a series of
treatments.
[0429] For antibodies, the preferred dosage is 0.1 mg/kg of body
weight (generally 10 mg/kg to 20 mg/kg). If the antibody is to act
in the brain, a dosage of 50 mg/kg to 100 mg/kg is usually
appropriate. Generally, partially human antibodies and fully human
antibodies have a longer half-life within the human body than other
antibodies. Accordingly, lower dosages and less frequent
administration is often possible. Modifications such as lipidation
can be used to stabilize antibodies and to enhance uptake and
tissue penetration (e.g., into the brain). A method for lipidation
of antibodies is described by Cruikshank et al. ((1997) J. Acquired
Immune Deficiency Syndromes and Human Retrovirology 14:193).
[0430] The present invention encompasses agents which modulate
expression or activity. An agent may, for example, be a small
molecule. For example, such small molecules include, but are not
limited to, peptides, peptidomimetics (e.g., peptoids), amino
acids, amino acid analogs, polynucleotides, polynucleotide analogs,
nucleotides, nucleotide analogs, organic or inorganic compounds
(i.e.,. including heteroorganic and organometallic compounds)
having a molecular weight less than about 10,000 grams per mole,
organic or inorganic compounds having a molecular weight less than
about 5,000 grams per mole, organic or inorganic compounds having a
molecular weight less than about 1,000 grams per mole, organic or
inorganic compounds having a molecular weight less than about 500
grams per mole, and salts, esters, and other pharmaceutically
acceptable forms of such compounds.
[0431] Exemplary doses include milligram or microgram amounts of
the small molecule per kilogram of subject or sample weight (e.g.,
about 1 microgram per kilogram to about 500 milligrams per
kilogram, about 100 micrograms per kilogram to about 5 milligrams
per kilogram, or about 1 microgram per kilogram to about 50
micrograms per kilogram. It is furthermore understood that
appropriate doses of a small molecule depend upon the potency of
the small molecule with respect to the expression or activity to be
modulated. When one or more of these small molecules is to be
administered to an animal (e.g., a human) in order to modulate
expression or activity of a polypeptide or nucleic acid of the
invention, a physician, veterinarian, or researcher may, for
example, prescribe a relatively low dose at first, subsequently
increasing the dose until an appropriate response is obtained. In
addition, it is understood that the specific dose level for any
particular animal subject will depend upon a variety of factors
including the activity of the specific compound employed, the age,
body weight, general health, gender, and diet of the subject, the
time of administration, the route of administration, the rate of
excretion, any drug combination, and the degree of expression or
activity to be modulated.
[0432] An antibody (or fragment thereof) may be conjugated to a
therapeutic moiety such as a cytotoxin, a therapeutic agent or a
radioactive ion. A cytotoxin or cytotoxic agent includes any agent
that is detrimental to cells. Examples include taxol, cytochalasin
B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,
tenoposide, vincristine, vinblastine, colchicin, doxorubicin,
daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,
actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,
tetracaine, lidocaine, propranolol, puromycin, maytansinoids, e.g.,
maytansinol (see U.S. Pat. No. 5,208,020), CC-1065 (see U.S. Pat.
Nos. 5,475,092, 5,585,499, 5,846,545) and analogs or homologs
thereof. Therapeutic agents include, but are not limited to,
antimetabolites (e.g., methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating
agents (e.g., mechlorethamine, thioepa chlorambucil, CC-1065,
melphalan, carmustine (BSNU) and lomustine (CCNU),
cyclothosphamide, busulfan, dibromomannitol, streptozotocin,
mitomycin C, and cis-dichlorodiarnine platinum (II) (DDP)
cisplatin), anthracyclines (e.g., daunorubicin (formerly
daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin
(formerly actinomycin), bleomycin, mithramycin, and anthramycin
(AMC)), and anti-mitotic agents (e.g., vincristine, vinblastine,
taxol and maytansinoids). Radioactive ions include, but are not
limited to iodine, yttrium and praseodymium.
[0433] The conjugates of the invention can be used for modifying a
given biological response, the drug moiety is not to be construed
as limited to classical chemical therapeutic agents. For example,
the drug moiety may be a protein or polypeptide possessing a
desired biological activity. Such proteins may include, for
example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or
diphtheria toxin; a protein such as tumor necrosis factor,
.alpha.-interferon, .beta.-interferon, nerve growth factor,
platelet derived growth factor, tissue plasminogen activator; or,
biological response modifiers such as, for example, lymphokines,
interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6
("IL-6"), granulocyte macrophase colony stimulating factor
("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or
other growth factors.
[0434] Alternatively, an antibody can be conjugated to a second
antibody to form an antibody heteroconjugate as described by Segal
in U.S. Pat. No. 4,676,980.
[0435] The nucleic acid molecules of the invention can be inserted
into vectors and used as gene therapy vectors. Gene therapy vectors
can be delivered to a subject by, for example, intravenous
injection, local administration (see U.S. Pat. 5,328,470) or by
stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl.
Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the
gene therapy vector can include the gene therapy vector in an
acceptable diluent, or can comprise a slow release matrix in which
the gene delivery vehicle is imbedded. Alternatively, where the
complete gene delivery vector can be produced intact from
recombinant cells, e.g., retroviral vectors, the pharmaceutical
preparation can include one or more cells which produce the gene
delivery system.
[0436] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0437] Methods of Treatment
[0438] The present invention provides for both prophylactic and
therapeutic methods of treating a subject at risk of (or
susceptible to) a disorder or having a disorder associated with
aberrant or unwanted 279 expression or activity. As used herein,
the term "treatment" is defined as the application or
administration of a therapeutic agent to a patient, or application
or administration of a therapeutic agent to an isolated tissue or
cell line from a patient, who has a disease, a symptom of disease
or a predisposition toward a disease, with the purpose to cure,
heal, alleviate, relieve, alter, remedy, ameliorate, improve or
affect the disease, the symptoms of disease or the predisposition
toward disease. A therapeutic agent includes, but is not limited
to, small molecules, peptides, antibodies, ribozymes and antisense
oligonucleotides.
[0439] With regards to both prophylactic and therapeutic methods of
treatment, such treatments may be specifically tailored or
modified, based on knowledge obtained from the field of
pharmacogenomics. "Pharmacogenomics", as used herein, refers to the
application of genomics technologies such as gene sequencing,
statistical genetics, and gene expression analysis to drugs in
clinical development and on the market. More specifically, the term
refers the study of how a patient's genes determine his or her
response to a drug (e.g., a patient's "drug response phenotype", or
"drug response genotype".) Thus, another aspect of the invention
provides methods for tailoring an individual's prophylactic or
therapeutic treatment with either the 279 molecules of the present
invention or 279 modulators according to that individual's drug
response genotype. Pharmacogenomics allows a clinician or physician
to target prophylactic or therapeutic treatments to patients who
will most benefit from the treatment and to avoid treatment of
patients who will experience toxic drug-related side effects.
[0440] In one aspect, the invention provides a method for
preventing in a subject, a disease or condition associated with an
aberrant or unwanted 279 expression or activity, by administering
to the subject a 279 or an agent which modulates 279 expression or
at least one 279 activity. Subjects at risk for a disease which is
caused or contributed to by aberrant or unwanted 279 expression or
activity can be identified by, for example, any or a combination of
diagnostic or prognostic assays as described herein. Administration
of a prophylactic agent can occur prior to the manifestation of
symptoms characteristic of the 279 aberrance, such that a disease
or disorder is prevented or, alternatively, delayed in its
progression. Depending on the type of 279 aberrance, for example, a
279, 279 agonist or 279 antagonist agent can be used for treating
the subject. The appropriate agent can be determined based on
screening assays described herein.
[0441] It is possible that some 279 disorders can be caused, at
least in part, by an abnormal level of gene product, or by the
presence of a gene product exhibiting abnormal activity. As such,
the reduction in the level and/or activity of such gene products
would bring about the amelioration of disorder symptoms.
[0442] As the 279 mRNA is expressed in the liver and at lower
levels in neural tissues, the molecules of the invention can be
used therapeutically and diagnostically to treat and/or diagnose
disorders involving aberrant activities of those cells.
[0443] Neural disorders include, but are not limited to, disorders
involving neurons, and disorders involving glia, such as
astrocytes, oligodendrocytes, ependymal cells, and microglia;
cerebral edema, raised intracranial pressure and herniation, and
hydrocephalus; malformations and developmental diseases, such as
neural tube defects, forebrain anomalies, posterior fossa
anomalies, and syringomyelia and hydromyelia; perinatal brain
injury; cerebrovascular diseases, such as those related to hypoxia,
ischemia, and infarction, including hypotension, hypoperfusion, and
low-flow states--global cerebral ischemia and focal cerebral
ischemia--infarction from obstruction of local blood supply,
intracranial hemorrhage, including intracerebral (intraparenchymal)
hemorrhage, subarachnoid hemorrhage and ruptured berry aneurysms,
and vascular malformations, hypertensive cerebrovascular disease,
including lacunar infarcts, slit hemorrhages, and hypertensive
encephalopathy; infections, such as acute meningitis, including
acute pyogenic (bacterial) meningitis and acute aseptic (viral)
meningitis, acute focal suppurative infections, including brain
abscess, subdural empyema, and extradural abscess, chronic
bacterial meningoencephalitis, including tuberculosis and
mycobacterioses, neurosyphilis, and neuroborreliosis (Lyme
disease), viral meningoencephalitis, including arthropod-borne
(Arbo) viral encephalitis, Herpes simplex virus Type 1, Herpes
simplex virus Type 2, Varicalla-zoster virus (Herpes zoster),
cytomegalovirus, poliomyelitis, rabies, and human immunodeficiency
virus 1, including HIV-1 meningoencephalitis (subacute
encephalitis), vacuolar myelopathy, AIDS-associated myopathy,
peripheral neuropathy, and AIDS in children, progressive multifocal
leukoencephalopathy, subacute sclerosing panencephalitis, fungal
meningoencephalitis, other infectious diseases of the nervous
system; transmissible spongiform encephalopathies (prion diseases);
demyelinating diseases, including multiple sclerosis, multiple
sclerosis variants, acute disseminated encephalomyelitis and acute
necrotizing hemorrhagic encephalomyelitis, and other diseases with
demyelination; degenerative diseases, such as degenerative diseases
affecting the cerebral cortex, including Alzheimer disease and Pick
disease, degenerative diseases of basal ganglia and brain stem,
including Parkinsonism, idiopathic Parkinson disease (paralysis
agitans), progressive supranuclear palsy, corticobasal degenration,
multiple system atrophy, including striatonigral degenration,
Shy-Drager syndrome, and olivopontocerebellar atrophy, and
Huntington disease; spinocerebellar degenerations, including
spinocerebellar ataxias, including Friedreich ataxia, and
ataxia-telanglectasia, degenerative diseases affecting motor
neurons, including amyotrophic lateral sclerosis (motor neuron
disease), bulbospinal atrophy (Kennedy syndrome), and spinal
muscular atrophy; inborn errors of metabolism, such as
leukodystrophies, including Krabbe disease, metachromatic
leukodystrophy, adrenoleukodystrophy, Pelizaeus-Merzbacher disease,
and Canavan disease, mitochondrial encephalomyopathies, including
Leigh disease and other mitochondrial encephalomyopathies; toxic
and acquired metabolic diseases, including vitamin deficiencies
such as thiamine (vitamin B1) deficiency and vitamin B12
deficiency, neurologic sequelae of metabolic disturbances,
including hypoglycemia, hyperglycemia, and hepatic encephatopathy,
toxic disorders, including carbon monoxide, methanol, ethanol, and
radiation, including combined methotrexate and radiation-induced
injury; tumors, such as gliomas, including astrocytoma, including
fibrillary (diffuse) astrocytoma and glioblastoma multiforme,
pilocytic astrocytoma, pleomorphic xanthoastrocytoma, and brain
stem glioma, oligodendroglioma, and ependymoma and related
paraventricular mass lesions, neuronal tumors, poorly
differentiated neoplasms, including medulloblastoma, other
parenchymal tumors, including primary brain lymphoma, germ cell
tumors, and pineal parenchymal tumors, meningiomas, metastatic
tumors, paraneoplastic syndromes, peripheral nerve sheath tumors,
including schwannoma, neurofibroma, and malignant peripheral nerve
sheath tumor (malignant schwannoma), and neurocutaneous syndromes
(phakomatoses), including neurofibromotosis, including Type 1
neurofibromatosis (NF1) and TYPE 2 neurofibromatosis (NF2),
tuberous sclerosis, and Von Hippel-Lindau disease.
[0444] Disorders which may be treated or diagnosed by methods
described herein include, but are not limited to, disorders
associated with an accumulation in the liver of fibrous tissue,
such as that resulting from an imbalance between production and
degradation of the extracellular matrix accompanied by the collapse
and condensation of preexisting fibers. The methods described
herein can be used to diagnose or treat hepatocellular necrosis or
injury induced by a wide variety of agents including processes
which disturb homeostasis, such as an inflammatory process, tissue
damage resulting from toxic injury or altered hepatic blood flow,
and infections (e.g., bacterial, viral and parasitic). For example,
the methods can be used for the early detection of hepatic injury,
such as portal hypertension or hepatic fibrosis. In addition, the
methods can be employed to detect liver fibrosis attributed to
inborn errors of metabolism, for example, fibrosis resulting from a
storage disorder such as Gaucher's disease (lipid abnormalities) or
a glycogen storage disease, A1-antitrypsin deficiency; a disorder
mediating the accumulation (e.g., storage) of an exogenous
substance, for example, hemochromatosis (iron-overload syndrome)
and copper storage diseases (Wilson's disease), disorders resulting
in the accumulation of a toxic metabolite (e.g., tyrosinemia,
fructosemia and galactosemia) and peroxisomal disorders (e.g.,
Zellweger syndrome). Additionally, the methods described herein may
be useful for the early detection and treatment of liver injury
associated with the administration of various chemicals or drugs,
such as for example, methotrexate, isonizaid, oxyphenisatin,
methyldopa, chlorpromazine, tolbutamide or alcohol, or which
represents a hepatic manifestation of a vascular disorder such as
obstruction of either the intrahepatic or extrahepatic bile flow or
an alteration in hepatic circulation resulting, for example, from
chronic heart failure, veno-occlusive disease, portal vein
thrombosis or Budd-Chiari syndrome.
[0445] As discussed, successful treatment of 279 disorders can be
brought about by techniques that serve to inhibit the expression or
activity of target gene products. For example, compounds, e.g., an
agent identified using an assays described above, that proves to
exhibit negative modulatory activity, can be used in accordance
with the invention to prevent and/or ameliorate symptoms of 279
disorders. Such molecules can include, but are not limited to
peptides, phosphopeptides, small organic or inorganic molecules, or
antibodies (including, for example, polyclonal, monoclonal,
humanized, anti-idiotypic, chimeric or single chain antibodies, and
Fab, F(ab').sub.2 and Fab expression library fragments, scFV
molecules, and epitope-binding fragments thereof).
[0446] Further, antisense and ribozyme molecules that inhibit
expression of the target gene can also be used in accordance with
the invention to reduce the level of target gene expression, thus
effectively reducing the level of target gene activity. Still
further, triple helix molecules can be utilized in reducing the
level of target gene activity. Antisense, ribozyme and triple helix
molecules are discussed above.
[0447] It is possible that the use of antisense, ribozyme, and/or
triple helix molecules to reduce or inhibit mutant gene expression
can also reduce or inhibit the transcription (triple helix) and/or
translation (antisense, ribozyme) of mRNA produced by normal target
gene alleles, such that the concentration of normal target gene
product present can be lower than is necessary for a normal
phenotype. In such cases, nucleic acid molecules that encode and
express target gene polypeptides exhibiting normal target gene
activity can be introduced into cells via gene therapy method.
Alternatively, in instances in that the target gene encodes an
extracellular protein, it can be preferable to co-administer normal
target gene protein into the cell or tissue in order to maintain
the requisite level of cellular or tissue target gene activity.
[0448] Another method by which nucleic acid molecules may be
utilized in treating or preventing a disease characterized by 279
expression is through the use of aptamer molecules specific for 279
protein. Aptamers are nucleic acid molecules having a tertiary
structure which permits them to specifically bind to protein
ligands (see, e.g., Osborne, et al. (1997) Curr. Opin. Chem Biol.
1: 5-9; and Patel, D. J. (1997) Curr Opin Chem Biol 1:32-46). Since
nucleic acid molecules may in many cases be more conveniently
introduced into target cells than therapeutic protein molecules may
be, aptamers offer a method by which 279 protein activity may be
specifically decreased without the introduction of drugs or other
molecules which may have pluripotent effects.
[0449] Antibodies can be generated that are both specific for
target gene product and that reduce target gene product activity.
Such antibodies may, therefore, by administered in instances
whereby negative modulatory techniques are appropriate for the
treatment of 279 disorders. For a description of antibodies, see
the Antibody section above.
[0450] In circumstances wherein injection of an animal or a human
subject with a 279 protein or epitope for stimulating antibody
production is harmful to the subject, it is possible to generate an
immune response against 279 through the use of anti-idiotypic
antibodies (see, for example, Herlyn, D. (1999) Ann Med 31:66-78;
and Bhattacharya-Chatterjee, M., and Foon, K. A. (1998) Cancer
Treat Res. 94:51-68). If an anti-idiotypic antibody is introduced
into a mammal or human subject, it should stimulate the production
of anti-anti-idiotypic antibodies, which should be specific to the
279 protein. Vaccines directed to a disease characterized by 279
expression may also be generated in this fashion.
[0451] In instances where the target antigen is intracellular and
whole antibodies are used, internalizing antibodies may be
preferred. Lipofectin or liposomes can be used to deliver the
antibody or a fragment of the Fab region that binds to the target
antigen into cells. Where fragments of the antibody are used, the
smallest inhibitory fragment that binds to the target antigen is
preferred. For example, peptides having an amino acid sequence
corresponding to the Fv region of the antibody can be used.
Alternatively, single chain neutralizing antibodies that bind to
intracellular target antigens can also be administered. Such single
chain antibodies can be administered, for example, by expressing
nucleotide sequences encoding single-chain antibodies within the
target cell population (see e.g., Marasco et al. (1993) Proc. Natl.
Acad. Sci. USA 90:7889-7893).
[0452] The identified compounds that inhibit target gene
expression, synthesis and/or activity can be administered to a
patient at therapeutically effective doses to prevent, treat or
ameliorate 279 disorders. A therapeutically effective dose refers
to that amount of the compound sufficient to result in amelioration
of symptoms of the disorders. Toxicity and therapeutic efficacy of
such compounds can be determined by standard pharmaceutical
procedures as described above.
[0453] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED.sub.50 with
little or no toxicity. The dosage can vary within this range
depending upon the dosage form employed and the route of
administration utilized. For any compound used in the method of the
invention, the therapeutically effective dose can be estimated
initially from cell culture assays. A dose can be formulated in
animal models to achieve a circulating plasma concentration range
that includes the IC.sub.50 (i.e., the concentration of the test
compound that achieves a half-maximal inhibition of symptoms) as
determined in cell culture. Such information can be used to more
accurately determine useful doses in humans. Levels in plasma can
be measured, for example, by high performance liquid
chromatography.
[0454] Another example of determination of effective dose for an
individual is the ability to directly assay levels of "free" and
"bound" compound in the serum of the test subject. Such assays may
utilize antibody mimics and/or "biosensors" that have been created
through molecular imprinting techniques. The compound which is able
to modulate 279 activity is used as a template, or "imprinting
molecule", to spatially organize polymerizable monomers prior to
their polymerization with catalytic reagents. The subsequent
removal of the imprinted molecule leaves a polymer matrix which
contains a repeated "negative image" of the compound and is able to
selectively rebind the molecule under biological assay conditions.
A detailed review of this technique can be seen in Ansell, R. J. et
al (1996) Current Opinion in Biotechnology 7:89-94 and in Shea, K.
J. (1994) Trends in Polymer Science 2:166-173. Such "imprinted"
affinity matrixes are amenable to ligand-binding assays, whereby
the immobilized monoclonal antibody component is replaced by an
appropriately imprinted matrix. An example of the use of such
matrixes in this way can be seen in Vlatakis, G. et al (1993)
Nature 361:645-647. Through the use of isotope-labeling, the "free"
concentration of compound which modulates the expression or
activity of 279 can be readily monitored and used in calculations
of IC.sub.50.
[0455] Such "imprinted" affinity matrixes can also be designed to
include fluorescent groups whose photon-emitting properties
measurably change upon local and selective binding of target
compound. These changes can be readily assayed in real time using
appropriate fiberoptic devices, in turn allowing the dose in a test
subject to be quickly optimized based on its individual IC.sub.50.
An rudimentary example of such a "biosensor" is discussed in Kriz,
D. et al (1995) Analytical Chemistry 67:2142-2144.
[0456] Another aspect of the invention pertains to methods of
modulating 279 expression or activity for therapeutic purposes.
Accordingly, in an exemplary embodiment, the modulatory method of
the invention involves contacting a cell with a 279 or agent that
modulates one or more of the activities of 279 protein activity
associated with the cell. An agent that modulates 279 protein
activity can be an agent as described herein, such as a nucleic
acid or a protein, a naturally-occurring target molecule of a 279
protein (e.g., a 279 substrate or receptor), a 279 antibody, a 279
agonist or antagonist, a peptidomimetic of a 279 agonist or
antagonist, or other small molecule.
[0457] In one embodiment, the agent stimulates one or 279
activities. Examples of such stimulatory agents include active 279
protein and a nucleic acid molecule encoding 279. In another
embodiment, the agent inhibits one or more 279 activities. Examples
of such inhibitory agents include antisense 279 nucleic acid
molecules, anti-279 antibodies, and 279 inhibitors. These
modulatory methods can be performed in vitro (e.g., by culturing
the cell with the agent) or, alternatively, in vivo (e.g., by
administering the agent to a subject). As such, the present
invention provides methods of treating an individual afflicted with
a disease or disorder characterized by aberrant or unwanted
expression or activity of a 279 protein or nucleic acid molecule.
In one embodiment, the method involves administering an agent
(e.g., an agent identified by a screening assay described herein),
or combination of agents that modulates (e.g., up regulates or down
regulates) 279 expression or activity. In another embodiment, the
method involves administering a 279 protein or nucleic acid
molecule as therapy to compensate for reduced, aberrant, or
unwanted 279 expression or activity.
[0458] Stimulation of 279 activity is desirable in situations in
which 279 is abnormally downregulated and/or in which increased 279
activity is likely to have a beneficial effect. For example,
stimulation of 279 activity is desirable in situations in which a
279 is downregulated and/or in which increased 279 activity is
likely to have a beneficial effect. Likewise, inhibition of 279
activity is desirable in situations in which 279 is abnormally
upregulated and/or in which decreased 279 activity is likely to
have a beneficial effect.
[0459] Pharmacogenomics
[0460] The 279 molecules of the present invention, as well as
agents, or modulators which have a stimulatory or inhibitory effect
on 279 activity (e.g., 279 gene expression) as identified by a
screening assay described herein can be administered to individuals
to treat (prophylactically or therapeutically) 279 associated
disorders (e.g., angiogenesis related disorders such as cancer,
cardiovascular disorders such as atherosclerosis and disorders
involving vascular tone) associated with aberrant or unwanted 279
activity. In conjunction with such treatment, pharmacogenomics
(i.e., the study of the relationship between an individual's
genotype and that individual's response to a foreign compound or
drug) may be considered. Differences in metabolism of therapeutics
can lead to severe toxicity or therapeutic failure by altering the
relation between dose and blood concentration of the
pharmacologically active drug. Thus, a physician or clinician may
consider applying knowledge obtained in relevant pharmacogenomics
studies in determining whether to administer a 279 molecule or 279
modulator as well as tailoring the dosage and/or therapeutic
regimen of treatment with a 279 molecule or 279 modulator.
[0461] Pharmacogenomics deals with clinically significant
hereditary variations in the response to drugs due to altered drug
disposition and abnormal action in affected persons. See, for
example, Eichelbaum, M. et al. (1996) Clin. Exp. Pharmacol.
Physiol. 23:983-985 and Linder, M. W. et al. (1997) Clin. Chem.
43:254-266. In general, two types of pharmacogenetic conditions can
be differentiated. Genetic conditions transmitted as a single
factor altering the way drugs act on the body (altered drug action)
or genetic conditions transmitted as single factors altering the
way the body acts on drugs (altered drug metabolism). These
pharmacogenetic conditions can occur either as rare genetic defects
or as naturally-occurring polymorphisms. For example,
glucose-6-phosphate dehydrogenase deficiency (G6PD) is a common
inherited enzymopathy in which the main clinical complication is
haemolysis after ingestion of oxidant drugs (anti-malarials,
sulfonamides, analgesics, nitrofurans) and consumption of fava
beans.
[0462] One pharmacogenomics approach to identifying genes that
predict drug response, known as "a genome-wide association", relies
primarily on a high-resolution map of the human genome consisting
of already known gene-related markers (e.g., a "bi-allelic" gene
marker map which consists of 60,000-100,000 polymorphic or variable
sites on the human genome, each of which has two variants.) Such a
high-resolution genetic map can be compared to a map of the genome
of each of a statistically significant number of patients taking
part in a Phase II/III drug trial to identify markers associated
with a particular observed drug response or side effect.
Alternatively, such a high resolution map can be generated from a
combination of some ten-million known single nucleotide
polymorphisms (SNPs) in the human genome. As used herein, a "SNP"
is a common alteration that occurs in a single nucleotide base in a
stretch of DNA. For example, a SNP may occur once per every 1000
bases of DNA. A SNP may be involved in a disease process, however,
the vast majority may not be disease-associated. Given a genetic
map based on the occurrence of such SNPs, individuals can be
grouped into genetic categories depending on a particular pattern
of SNPs in their individual genome. In such a manner, treatment
regimens can be tailored to groups of genetically similar
individuals, taking into account traits that may be common among
such genetically similar individuals.
[0463] Alternatively, a method termed the "candidate gene
approach," can be utilized to identify genes that predict drug
response. According to this method, if a gene that encodes a drug's
target is known (e.g., a 279 protein of the present invention), all
common variants of that gene can be fairly easily identified in the
population and it can be determined if having one version of the
gene versus another is associated with a particular drug
response.
[0464] Alternatively, a method termed the "gene expression
profiling," can be utilized to identify genes that predict drug
response. For example, the gene expression of an animal dosed with
a drug (e.g., a 279 molecule or 279 modulator of the present
invention) can give an indication whether gene pathways related to
toxicity have been turned on.
[0465] Information generated from more than one of the above
pharmacogenomics approaches can be used to determine appropriate
dosage and treatment regimens for prophylactic or therapeutic
treatment of an individual. This knowledge, when applied to dosing
or drug selection, can avoid adverse reactions or therapeutic
failure and thus enhance therapeutic or prophylactic efficiency
when treating a subject with a 279 molecule or 279 modulator, such
as a modulator identified by one of the exemplary screening assays
described herein.
[0466] The present invention further provides methods for
identifying new agents, or combinations, that are based on
identifying agents that modulate the activity of one or more of the
gene products encoded by one or more of the 279 genes of the
present invention, wherein these products may be associated with
resistance of the cells to a therapeutic agent. Specifically, the
activity of the proteins encoded by the 279 genes of the present
invention can be used as a basis for identifying agents for
overcoming agent resistance. By blocking the activity of one or
more of the resistance proteins, target cells, e.g., human cells,
will become sensitive to treatment with an agent that the
unmodified target cells were resistant to.
[0467] Monitoring the influence of agents (e.g., drugs) on the
expression or activity of a 279 protein can be applied in clinical
trials. For example, the effectiveness of an agent determined by a
screening assay as described herein to increase 279 gene
expression, protein levels, or upregulate 279 activity, can be
monitored in clinical trials of subjects exhibiting decreased 279
gene expression, protein levels, or downregulated 279 activity.
Alternatively, the effectiveness of an agent determined by a
screening assay to decrease 279 gene expression, protein levels, or
downregulate 279 activity, can be monitored in clinical trials of
subjects exhibiting increased 279 gene expression, protein levels,
or upregulated 279 activity. In such clinical trials, the
expression or activity of a 279 gene, and preferably, other genes
that have been implicated in, for example, a 279-associated
disorder can be used as a "read out" or markers of the phenotype of
a particular cell.
[0468] 279 Informatics
[0469] The sequence of a 279 molecule is provided in a variety of
media to facilitate use thereof. A sequence can be provided as a
manufacture, other than an isolated nucleic acid or amino acid
molecule, which contains a 279. Such a manufacture can provide a
nucleotide or amino acid sequence, e.g., an open reading frame, in
a form which allows examination of the manufacture using means not
directly applicable to examining the nucleotide or amino acid
sequences, or a subset thereof, as they exists in nature or in
purified form. The sequence information can include, but is not
limited to, 279 full-length nucleotide and/or amino acid sequences,
partial nucleotide and/or amino acid sequences, polymorphic
sequences including single nucleotide polymorphisms (SNPs), epitope
sequence, and the like. In a preferred embodiment, the manufacture
is a machine-readable medium, e.g., a magnetic, optical, chemical
or mechanical information storage device.
[0470] As used herein, "machine-readable media" refers to any
medium that can be read and accessed directly by a machine, e.g., a
digital computer or analogue computer. Non-limiting examples of a
computer include a desktop PC, laptop, mainframe, server (e.g., a
web server, network server, or server farm), handheld digital
assistant, pager, mobile telephone, and the like. The computer can
be stand-alone or connected to a communications network, e.g., a
local area network (such as a VPN or intranet), a wide area network
(e.g., an Extranet or the Internet), or a telephone network (e.g.,
a wireless, DSL, or ISDN network). Machine-readable media include,
but are not limited to: magnetic storage media, such as floppy
discs, hard disc storage medium, and magnetic tape; optical storage
media such as CD-ROM; electrical storage media such as RAM, ROM,
EPROM, EEPROM, flash memory, and the like; and hybrids of these
categories such as magnetic/optical storage media.
[0471] A variety of data storage structures are available to a
skilled artisan for creating a machine-readable medium having
recorded thereon a nucleotide or amino acid sequence of the present
invention. The choice of the data storage structure will generally
be based on the means chosen to access the stored information. In
addition, a variety of data processor programs and formats can be
used to store the nucleotide sequence information of the present
invention on computer readable medium. The sequence information can
be represented in a word processing text file, formatted in
commercially-available software such as WordPerfect and Microsoft
Word, or represented in the form of an ASCII file, stored in a
database application, such as DB2, Sybase, Oracle, or the like. The
skilled artisan can readily adapt any number of data processor
structuring formats (e.g., text file or database) in order to
obtain computer readable medium having recorded thereon the
nucleotide sequence information of the present invention.
[0472] In a preferred embodiment, the sequence information is
stored in a relational database (such as Sybase or Oracle). The
database can have a first table for storing sequence (nucleic acid
and/or amino acid sequence) information. The sequence information
can be stored in one field (e.g., a first column) of a table row
and an identifier for the sequence can be store in another field
(e.g., a second column) of the table row. The database can have a
second table, e.g., storing annotations. The second table can have
a field for the sequence identifier, a field for a descriptor or
annotation text (e.g., the descriptor can refer to a functionality
of the sequence, a field for the initial position in the sequence
to which the annotation refers, and a field for the ultimate
position in the sequence to which the annotation refers.
Non-limiting examples for annotation to nucleic acid sequences
include polymorphisms (e.g., SNP's) translational regulatory sites
and splice junctions. Non-limiting examples for annotations to
amino acid sequence include polypeptide domains, e.g., a domain
described herein; active sites and other functional amino acids;
and modification sites.
[0473] By providing the nucleotide or amino acid sequences of the
invention in computer readable form, the skilled artisan can
routinely access the sequence information for a variety of
purposes. For example, one skilled in the art can use the
nucleotide or amino acid sequences of the invention in computer
readable form to compare a target sequence or target structural
motif with the sequence information stored within the data storage
means. A search is used to identify fragments or regions of the
sequences of the invention which match a particular target sequence
or target motif. The search can be a BLAST search or other routine
sequence comparison, e.g., a search described herein.
[0474] Thus, in one aspect, the invention features a method of
analyzing 279, e.g., analyzing structure, function, or relatedness
to one or more other nucleic acid or amino acid sequences. The
method includes: providing a 279 nucleic acid or amino acid
sequence; comparing the 279 sequence with a second sequence, e.g.,
one or more preferably a plurality of sequences from a collection
of sequences, e.g., a nucleic acid or protein sequence database to
thereby analyze 279. The method can be performed in a machine,
e.g., a computer, or manually by a skilled artisan.
[0475] The method can include evaluating the sequence identity
between a 279 sequence and a database sequence. The method can be
performed by accessing the database at a second site, e.g., over
the Internet.
[0476] As used herein, a "target sequence" can be any DNA or amino
acid sequence of six or more nucleotides or two or more amino
acids. A skilled artisan can readily recognize that the longer a
target sequence is, the less likely a target sequence will be
present as a random occurrence in the database. Typical sequence
lengths of a target sequence are from about 10 to 100 amino acids
or from about 30 to 300 nucleotide residues. However, it is well
recognized that commercially important fragments, such as sequence
fragments involved in gene expression and protein processing, may
be of shorter length.
[0477] Computer software is publicly available which allows a
skilled artisan to access sequence information provided in a
computer readable medium for analysis and comparison to other
sequences. A variety of known algorithms are disclosed publicly and
a variety of commercially available software for conducting search
means are and can be used in the computer-based systems of the
present invention. Examples of such software include, but are not
limited to, MacPattern (EMBL), BLASTN and BLASTX (NCBI).
[0478] Thus, the invention features a method of making a computer
readable record of a sequence of a 279 sequence which includes
recording the sequence on a computer readable matrix. In a
preferred embodiment the record includes one or more of the
following: identification of an ORF; identification of a domain,
region, or site; identification of the start of transcription;
identification of the transcription terminator; the full length
amino acid sequence of the protein, or a mature form thereof; the
5' end of the translated region.
[0479] In another aspect, the invention features, a method of
analyzing a sequence. The method includes: providing a 279
sequence, or record, in machine-readable form; comparing a second
sequence to the 279 sequence; thereby analyzing a sequence.
Comparison can include comparing to sequences for sequence identity
or determining if one sequence is included within the other, e.g.,
determining if the 279 sequence includes a sequence being compared.
In a preferred embodiment the 279 or second sequence is stored on a
first computer, e.g., at a first site and the comparison is
performed, read, or recorded on a second computer, e.g., at a
second site. E.g., the 279 or second sequence can be stored in a
public or proprietary database in one computer, and the results of
the comparison performed, read, or recorded on a second computer.
In a preferred embodiment the record includes one or more of the
following: identification of an ORF; identification of a domain,
region, or site; identification of the start of transcription;
identification of the transcription terminator; the full length
amino acid sequence of the protein, or a mature form thereof; the
5' end of the translated region.
[0480] In another aspect, the invention provides a machine-readable
medium for holding instructions for performing a method for
determining whether a subject has a 279-associated disease or
disorder or a pre-disposition to a 279-associated disease or
disorder, wherein the method comprises the steps of determining 279
sequence information associated with the subject and based on the
279 sequence information, determining whether the subject has a
279-associated disease or disorder or a pre-disposition to a
279-associated disease or disorder and/or recommending a particular
treatment for the disease, disorder or pre-disease condition.
[0481] The invention further provides in an electronic system
and/or in a network, a method for determining whether a subject has
a 279-associated disease or disorder or a pre-disposition to a
disease associated with a 279 wherein the method comprises the
steps of determining 279 sequence information associated with the
subject, and based on the 279 sequence information, determining
whether the subject has a 279-associated disease or disorder or a
pre-disposition to a 279-associated disease or disorder, and/or
recommending a particular treatment for the disease, disorder or
pre-disease condition. In a preferred embodiment, the method
further includes the step of receiving information, e.g.,
phenotypic or genotypic information, associated with the subject
and/or acquiring,-from a network phenotypic information associated
with the subject. The information can be stored in a database,
e.g., a relational database. In another embodiment, the method
further includes accessing the database, e.g., for records relating
to other subjects, comparing the 279 sequence of the subject to the
279 sequences in the database to thereby determine whether the
subject as a 279-associated disease or disorder, or a
pre-disposition for such.
[0482] The present invention also provides in a network, a method
for determining whether a subject has a 279 associated disease or
disorder or a pre-disposition to a 279-associated disease or
disorder associated with 279, said method comprising the steps of
receiving 279 sequence information from the subject and/or
information related thereto, receiving phenotypic information
associated with the subject, acquiring information from the network
corresponding to 279 and/or corresponding to a 279-associated
disease or disorder (e.g., an angiogenesis-related disorders, such
as cancer, or a cardiovascular disorder, e.g., atherosclerosis),
and based on one or more of the phenotypic information, the 279
information (e.g., sequence information and/or information related
thereto), and the acquired information, determining whether the
subject has a 279-associated disease or disorder or a
pre-disposition to a 279-associated disease or disorder. The method
may further comprise the step of recommending a particular
treatment for the disease, disorder or pre-disease condition.
[0483] The present invention also provides a method for determining
whether a subject has a 279-associated disease or disorder or a
pre-disposition to a 279-associated disease or disorder, said
method comprising the steps of receiving information related to 279
(e.g., sequence information and/or information related thereto),
receiving phenotypic information associated with the subject,
acquiring information from the network related to 279 and/or
related to a 279-associated disease or disorder, and based on one
or more of the phenotypic information, the 279 information, and the
acquired information, determining whether the subject has a
279-associated disease or disorder or a pre-disposition to a
279-associated disease or disorder. The method may further comprise
the step of recommending a particular treatment for the disease,
disorder or pre-disease condition.
[0484] This invention is further illustrated by the following
examples that should not be construed as limiting. The contents of
all references, patents and published patent applications cited
throughout this application are incorporated herein by
reference.
EXAMPLES
Example 1
[0485] Identification and Characterization of Human 279 cDNA
[0486] The human 279 nucleic acid sequence is recited as
follows:
1 (SEQ ID NO:1) ATGGGCAACCACACGTGGGAGGGCTGCCACGTGGACTCGCGCG-
TGGACCA CCTCTTTCCGCCATCCCTCTACATCTTTGTCATCGGCGTGGGGCTGCCC- A
CCAACTGCCTGGCTCTGTGGGCGGCCTACCGCCAGGTGCAACAGCGCAAC
GAGCTGGGCGTCTACCTGATGAACCTCAGCATCGCCGACCTGCTGTACAT
CTGCACGCTGCCGCTGTGGGTGGACTACTTCCTGCACCACGACAACTGGA
TCCACGGCCCCGGGTCCTGCAAGCTCTTTGGGTTCATCTTCTACACCAAT
ATCTACATCAGCATCGCCTTCCTGTGCTGCATCTCGGTGGACCGCTACCT
GGCTGTGGCCCACCCACTCCGCTTCGCCCGCCTGCGCCGCGTCAAGACCG
CCGTGGCCGTGAGCTCCGTGGTCTGGGCCACGGAGCTGGGCGCCAACTCG
GCGCCCCTGTTCCATGACGAGCTCTTCCGAGACCGCTACAACCACACCTT
CTGCTTTGAGAAGTTCCCCATGGAAGGCTGGGTGGCCTGGATGAACCTCT
ATCGGGTGTTCGTGGGCTTCCTCTTCCCGTGGGCGCTCATGCTGCTGTCG
TACCGGGGCATCCTGCGGGCCGTGCGGGGCAGCGTGTCCACCGAGCGCCA
GGAGAAGGCCAAGATCAAGCGGCTGGCCCTCAGCCTCATCGCCATCGTGC
TGGTCTGCTTTGCGCCCTATCACGTGCTCTTGCTGTCCCGCAGCGCCATC
TACCTGGGCCGCCCCTGGGACTGCGGCTTCGAGGAGCGCGTCTTTTCTGC
ATACCACAGCTCACTGGCTTTCACCAGCCTCAACTGTGTGGCGGACCCCA
TCCTCTACTGCCTGGTCAACGAGGGCGCCCGCAGCGATGTGGCCAAGGCC
CTGCACAACCTGCTCCGCTTTCTGGCCAGCGACAAGCCCCAGGAGATGGC
CAATGCCTCGCTCACCCTGGAGACCCCACTCACCTCCAAGAGGAACAGCA
CAGCCAAAGCCATGACTGGCAGCTGGGCGGCCACTCCGCCCTCCCAGGGG
GACCAGGTGCAGCTGAAGATGCTGCCGCCAGCACAATGA.
[0487] The human 279 sequence (FIG. 1; SEQ ID NO: 1), which is
approximately 1089 nucleotides long. The nucleic acid sequence
includes an initiation codon (ATG) and a termination codon (TAA)
which are underscored above. The region between and inclusive of
the initiation codon and the termination codon is a
methionine-initiated coding sequence of about 1089 nucleotides,
including the termination codon (nucleotides indicated as "coding"
of SEQ ID NO: 1; SEQ ID NO: 3). The coding sequence encodes a 362
amino acid protein (SEQ ID NO: 2), which is recited as follows:
2 (SEQ ID NO:2) MGNHTWEGCHVDSRVDHLFPPSLYIFVIGVGLPTNCLALWAAY-
RQVQQRN ELGVYLMNLSIADLLYICTLPLWVDYFLHHDNWIHGPGSCKLFGFIFYT- N
IYISIAFLCCISVDRYLAVAHPLRFARLRRVKTAVAVSSVVWATELGANS
APLFHDELFRDRYNHTFCFEKFPMEGWVAWMNLYRVFVGFLFPWALMLLS
YRGILRAVRGSVSTERQEKAKIKRLALSLIAIVLVCFAPYHVLLLSRSAI
YLGRPWDCGFEERVFSAYHSSLAFTSLNCVADPILYCLVNEGARSDVAKA
LHNLLRFLASDKPQEMANASLTLETPLTSKRNSTAKAMTGSWAATPPSQG
DQVQLKMLPPAQ.
Example 2
[0488] Tissue Distribution of 279 mRNA by TaqMan Analysis
[0489] Endogenous human 279 gene expression was determined using
the Perkin-Elmer/ABI 7700 Sequence Detection System which employs
TaqMan technology. Briefly, TaqMan technology relies on standard
RT-PCR with the addition of a third gene-specific oligonucleotide
(referred to as a probe) which has a fluorescent dye coupled to its
5' end (typically 6-FAM) and a quenching dye at the 3' end
(typically TAMRA). When the fluorescently tagged oligonucleotide is
intact, the fluorescent signal from the 5' dye is quenched. As PCR
proceeds, the 5' to 3' nucleolytic activity of Taq polymerase
digests the labeled primer, producing a free nucleotide labeled
with 6-FAM, which is now detected as a fluorescent signal. The PCR
cycle where fluorescence is first released and detected is directly
proportional to the starting amount of the gene of interest in the
test sample, thus providing a quantitative measure of the initial
template concentration. Samples can be internally controlled by the
addition of a second set of primers/probe specific for a
housekeeping gene such as GAPDH which has been labeled with a
different fluorophore on the 5' end (typically VIC).
[0490] To determine the level of 279 in various human tissues a
primer/probe set was designed. Total RNA was prepared from a series
of human tissues using an RNeasy kit from Qiagen. First strand cDNA
was prepared from 1 .mu.g total RNA using an oligo-dT primer and
Superscript II reverse transcriptase (Gibco/BRL). cDNA obtained
from approximately 50 ng total RNA was used per TaqMan
reaction.
3TABLE 1 Expression of 279 mRNA in human tissues Tissue Type
Expression Artery normal 10.7092 Aorta diseased 3.5573 Vein normal
0.1605 Coronary SMC 42.6888 HUVEC 83.9108 Hemangioma 17.0392 Heart
normal 21.1969 Heart CHF 9.8204 Kidney 9.0054 Skeletal Muscle
5.8595 Adipose normal 2.0011 Pancreas 1.7121 primary osteoblasts 0
Osteoclasts (diff) 0 Skin normal 2.015 Spinal cord normal 4.0022
Brain Cortex normal 3.6955 Brain Hypothalamus normal 16.3451 Nerve
28.8557 DRG (Dorsal Root 7.34 Ganglion) Breast normal 3.1949 Breast
tumor 0.7099 Ovary normal 1.4101 Ovary Tumor 0.18 Prostate Normal
0.8269 Prostate Tumor 1.043 Salivary glands 0.0444 Colon normal
0.1976 Colon Tumor 2.7336 Lung normal 2.3307 Lung tumor 1.1063 Lung
COPD 2.0573 Colon IBD 0.2399 Liver normal 1.791 Liver fibrosis
11.8006 Spleen normal 10.4888 Tonsil normal 0.1135 Lymph node
normal 0.681 Small intestine normal 0.6245 Skin-Decubitus 7.1641
Synovium 1.8287 BM-MNC 0.0155 Activated PBMC 0 Neutrophils 0.0146
Megakaryocytes 0 Erythroid 0
[0491] Table 1 depicts expression of 279 mRNA in a panel of human
tissues. Elevated expression is detected in cardiovascular tissues,
e.g., coronary smooth muscle cells (SMC), human umbilical vein
endothelial cells (HUVEC), normal and diseased (congestive heart
failure, CHF) heart, as well as endothelial cell-rich tissues, such
as hemangiomas. Lower levels of expression are detected in
neurological tissues, e.g., nerve, brain, and in the liver.
4TABLE 2 279 mRNA Expression in Cardiovascular Vessel Panel Tissue
Type Expression Aortic SMC 0.3537 Coronary SMC 75.363 Huvec Static
90.2456 Huvec LSS 65.1541 H/Adipose/MET 8 1.5009
H/Artery/Normal/Carotid/CLN 595 2.6588 H/Artery/Normal/Carotid/CL-
N 598 2.4213 H/Artery/normal/NDR 352 7.7585 H/Muscular
Artery/Normal/AMC 236 0.859 H/Muscular Artery/Normal/AMC 254/
0.4635 H/Muscular Artery/Normal/AMC 259 0.4603 H/Muscular
Artery/Normal/AMC 261 2.0933 H/Muscular Artery/Normal/AMC 275
0.6978 H/Aorta/Diseased/PIT 732 6.7308 H/Aorta/Diseased/PIT 710
1.9599 H/Aorta/Diseased/PIT 711 2.4297 H/Aorta/Diseased/PIT 712
5.582 H/Artery/Diseased/iliac/NDR 753 3.9334
H/Artery/Diseased/Tibial/PIT 679 4.5973 H/Vein/Normal/SaphenousAMC
107 0.4035 H/Vein/Normal/NDR 239 0.2893 H/Vein/Normal/Saphenous/NDR
237 2.3227 H/Vein/Normal/PIT 1010 0.7769 H/Vein/Normal/AMC 191
1.3526 H/Vein/Normal/AMC 130 1.1063 H/Vein/Normal/AMC 188 0.9112
H/Vein/Normal/AMC 196 0.2118 H/Vein/Normal/AMC 211 0
H/Vein/Normal/AMC 214 0.4385 M/Artery/Diseased/CAR 1174 2.9707
M/Artery/Diseased/CAR 1175 0.2626 M/Aorta/Normal/PRI 286 8.82
M/Artery/Normal/PRI 324 6.3457 M/Aorta/Normal/PRI 264 66.7541
M/Artery/Normal/PRI 320 4.03 M/Vein/Normal/PRI 328 6.3899 LCM NDR
352 0 LCM AMC 211 0 LCM AMC 196 0 HUVEC Vehicle 18.2621 HUVEC Mev
10.8212 HAEC Vehicle 30.8198 HAEC Mev 14.3779
[0492] Table 2 depicts 279 mRNA expression in human cardiovascular
tissues. Highest levels of expression of 279 mRNA were found in
heart and cardiovascular tissues, e.g., human umbilical vein
endothelial cells (static), human umbilical vein endothelial cells
(LSS), and the normal aorta. 279 mRNA expression was elevated in
cells treated with laminar shear stress, "LSS" and untreated
controls, called "static." The LSS paradigm is designed to simulate
blood flow in a straight unbranched stretch of artery (treatment is
10 dyn/cm.sup.2 LSS for 24 h). In vitro, it is known to increase
nitric oxide production, decrease adhesion molecule expression and
proliferation, and protect cells from the normal apoptotic response
to serum withdrawal.
5TABLE 3 ApoE Aorta Mouse Model of Atherosclerosis APOE 1 Mean RE 4
wk ApoE ab 18.7 4 wk ApoE ar 23.5 4 wk ApoE ar 23.1 4 wk ApoE ar
23.2 10 wk ApoE ab 21.7 10 wk ApoE ab 41.3 10 wk ApoE ab 48.7 10 wk
ApoE ab 26.2 10 wk ApoE ar 60.8 10 wk ApoE ar 85.9 10 wk ApoE ar
53.0 10 wk ApoE ar 32.0 12 wk ApoE ab 4.2 12 wk ApoE ar 10.7 15 wk
ApoE ab 30.1 15 wk ApoE ab 27.8 15 wk ApoE ar 130.0 15 wk ApoE ar
137.7 20 wk ApoE ab 50.9 20 wk ApoE ab 6.4 22 wk ApoE ab 7.6 20 wk
ApoE ab 30.2 20 wk ApoE ar 94.5 22 wk ApoE ar 20.1 20 wk ApoE ar
75.7 20 wk ApoE ar 61.0 25 wk ApoE ab 9.6 25 wk ApoE ar 54.3 30 wk
ApoE ab 8.0 30 wk ApoE ab 6.5 30 wk ApoE ab 9.9 30 wk ApoE ar 11.3
12 wk WT ar 10.6 24 wk WT ab 19.5 24 wk WT ar 46.5 17 wk ApoE ab
30.7 17 wk ApoE ar 41.1 10 wk ApoE ab 53.5 10 wk ApoE ar 52.6 12 wk
ApoE ar 4.6 20 wk ApoE ab 42.3 20 wk ApoE ar 28.9 20 wk ApoE ab
28.1 20 wk ApoE ar 20.3 30 wk ApoE ar 30 wk ApoE ar
[0493] Table 3 depicts progressive upregulation of 279 mRNA with
disease development. Marked upregulation is observed in the aortic
arch (where abundant atherosclerotic plaque development occurs)
relative to the abdominal aorta (where risk for plaque development
is low).
6TABLE 4 279 mRNA Expression in Cardiovascular Organ Panel Tissue
Type Expression H/Fetal Heart/normal/BWH 4 1.5975
H/Heart/Normal/Atrium/MPI 1097 46.3914 H/Heart/Normal/Ventricle/PIT
272 17.9484 H/Heart/Normal/Ventricle/PIT 206 52.3742
H/Heart/Normal/Ventricle- /PIT 204 63.5925
H/Heart/Normal/Ventricle/PIT 205 68.3934
H/Heart/Diseased/Ventricle/ELI 5 6.3899 H/Heart/Diseased/Ventricl-
e/PIT 16 1.7603 H/Heart/Diseased/Ventricle/PIT 1 3.1509
H/Heart/Diseased/Ventricle/PIT 14 6.3019 H/Kidney/normal/NDR 171
2.7431 H/Kidney/normal/NDR 179 1.8223 H/Kidney/normal/PIT 289
22.6397 H/Kidney/normal/PIT 351 4.5655 H/Kidney/normal/PIT 353
2.9095 H/Kidney/HT/NDR 233 1.1573 H/Kidney/HT/NDR 224 1.3811
H/Kidney/HT/CHT 1176 1.2023 H/Kidney/HT/NDR 252 5.3732
H/Kidney/HT/CHT 762 1.3387 H/Skeletal Muscle/Normal/PIT 915 2.1974
H/Skeletal Muscle/Normal/PIT 685 0 H/Skeletal Muscle/Normal/PIT 428
29.977 H/Liver/Normal/MPI 146 0.0385 H/Liver/Normal/CHT 339 0.2934
H/Liver/Normal/CHT 1237 0.1055 M/Heart/Normal/Ventricle/M- PI 96
142.5955 WF Liver Sample 420.4482 WF Liver Sample 1979.3133
[0494] Table 4 depicts expression of 279 in a human cardiovascular
panel. High expression of 279 mRNA is observed in the normal heart
(e.g., in the normal heart ventricle and atrium.
7TABLE 5 279 mRNA Expression in Angiogenesis Panel Tissue Type
Expression ONC 101 Hemangioma 23.1154 ONC 102 Hemangioma 23.357 ONC
103 Hemangioma 6.8961 TCH 002 Hemangioma 0 TCH 003 Hemangioma
3.3076 TCH 004 Hemangioma 3.1619 CHT 1273 Glioblastoma 2.4129 CHT
216 Glioblastoma 1.1857 CHT 501 Glioblastoma 0.5038 NDR 203 Normal
Kidney 2.8595 PIT 213 Renal Cell 0.7299 Carcinoma CHT 732 Wilms
Tumor 4.5183 CHT 765 Wilms Tumor 2.4046 NDR 295 Skin 0 CHT 1424
Uterine 0.8893 Adenocarcinoma CHT 1238 2.7621 Neuroblastoma BWH 78
Fetal Adrenal 1.2886 BWH 74 Fetal Kidney 8.8814 BWH 4 Fetal Heart
1.3526 MPI 849 Normal Heart 21.1236 CLN 746 Spinal cord 2.4722
[0495] Table 5 depicts high expression of 279 mRNA in hemangioma
and in the normal heart.
[0496] Changes in 279 gene expression were evaluated in a series of
angiogenic models. FIG. 3 is a bar graph depicting increased
expression of murine 279 mRNA in the developing mouse heart. In
rodents, there is major proliferation and development of the
cardiac blood vessels during the first three weeks after birth
(Tomanek, R J (1996) Cardiovasc Res. 31: E46-E51, and Olivetti, G
et al. (1980) Circ. Res. 46: 503-512). FIG. 3 is a panel that
includes pooled heart samples from several timepoints during the
period of vascular growth (5, 7, 10, 14, 17, 21 days after birth),
as well as the mothers of these newborns ("M", growth state of
vessels in heart is not well-defined) and normal adults ("A"), with
quiescent vasculature. Regulation during the newborn period, or
differential expression between newborns and adults supports an
angiogenic function for 279 protein.
[0497] FIG. 4 is a bar graph depicting 279 mRNA expression in a
superovulation model of angiogenesis. Hormone treatments induce the
synchronous maturation of multiple ovarian follicles and subsequent
superovulation in mice. Development of each follicle and resulting
corpus luteum is accompanied by robust angiogensis. Treatment
begins at puberty (25 days), when the background of normal
ovulation is minimal, but the mice are primed to respond to
hormone. Younger mice, without ongoing ovulation, serve as
low-angiogenesis controls. As mice age during the protocol and
normal ovulation begins, age matched control mice are used as
well.
[0498] Groups of control mice are represented by the five bars on
the left, labeled with ages. The four bars on the middle panel
represent groups of homonally treated mice. The treatment
includes:
[0499] 10 IU PMSG (pregnant mare serum gonadatrophin) injected
IP.
[0500] 10 IU PMSG (pregnant mare serum gonadatrophin) injected
IP.
[0501] 10 IU hCG (human chorionic gonadatrophin) injected IP.
[0502] 10 IU hCG (human chorionic gonadatrophin) injected IP.
[0503] Group O2 (first bar in the middle panel) received treatment
1, group O3 received 1 and 2, group O4 received 1, 2, and 3, and
group O5 received all four treatments. The superovulation markers
show regulation of marker genes. Pecam, Tie-2, Flk, and Flt are
endothelial cell markers and Flk and Flt (VEGF-receptors) may also
be upregulated in the growing vessels. Expression of these markers
peaks in group O4. VEGF, an angiogenesis inducer, peaks at the same
time. These results indicate that the peak of angiogenesis and
vessel density occurs in group O4.
[0504] After the peak is reached, ongoing angiogenesis is
eventually replaced by vessel regression. The four samples at the
night of the graph represent groups of mice that received all four
treatments, and were sacrificed at later timepoints during
regression. Ang2 is a regression marker, which peaks in groups OE
and OF. 279 mRNA shows a similar pattern of regulation as other
angiogenic markers, such as Pecam, Tie-2, FIk, Flt and VEGF, thus
further supporting a role of the 279 polypeptide in
pro-angiogenesis. Regulation similar to that of Ang2 supports
involvement in vascular regression. Depending on other factors,
Ang2 itself and possibly similarly regulated genes may ultimately
contribute to angiogenesis by destabilizing pre-existing vessels,
so function can be interpreted as pro- or anti-angiogenic.
[0505] FIG. 5 is a bar graph depicting expression/regulation of 279
mRNA in endothelial cells (ECs) isolated from the indicated tissues
and exposed to the indicated treatments. The bars at the left of
the panel are ECs treated with laminar shear stress, "LSS"
(2.sup.nd and 4.sup.th bars, pattern) and untreated controls,
called "static" (1.sup.st and 3.sup.rd bars, solid blue). The LSS
paradigm is designed to simulate blood flow in a straight
unbranched stretch of artery (treatment is 10 dyn/cm.sup.2 LSS for
24 h). In vitro, it is known to increase nitric oxide production,
decrease adhesion molecule expression and proliferation, and
protect cells from the normal apoptotic response to serum
withdrawal. Because of these effects in vitro, the paradigm is
believed to reproduce the athero-protected (relatively lesion-free)
state that is observed in straight, unbranched arteries
experiencing laminar shear stress in vivo. However, the real
correlation between the in vitro and in vivo situations is unclear.
Looking at known genes, there is not a consistent direction of
shear regulation that is correlated with pro- vs.
anti-atherosclerotic or angiogenic function.
[0506] The next five groups of samples shown in FIG. 5 cover
proliferation state, looking at ECs from three sources: Human
vascular endothelial cells (HUVECS), human microvesicular cells
(HMVEC) from cardiac and lung tissues, and aortic tissues. The bars
labeled as "Prolif" are rapidly proliferating cells in rich medium.
The solid bars labeled as "Conf" are confluent, contact-inhibited
cells in the same medium. The striped bars labeled "-GF" are
confluent, contact-inhibited cells deprived of growth factors. In
general, genes upregulated in the proliferating samples are
believed to have a pro-proliferation/pro-angiogenic function.
However, controls on proliferation are complex and clearly involve
feedback mechanisms. The fourth bar in the third group of samples
is cells treated with interleukin-1 beta (IL-1) at 34 ng/ml for 6
hours. The control sample is the solid bar in the same group (also
the confluent sample for that group). Regulation by IL-1 is
associated with atherosclerosis, and can also support an
angiogenenic role for the 279 polypeptide. The eighth group of
samples is a TNF-alpha treatment paradigm, also associated with
atherosclerosis and angiogenesis. The solid bars labeled as "C" are
controls. The second and fourth (patterned bars) samples were
treated with 10 ng/ml TNF for 4 and 14 h, respectively. The last
sample is 293 cells (black). These are human embryonic kidney
epithelial cells. They are used to compare the EC expression to a
non-EC sample.
[0507] FIG. 6 is a bar graph depicting 279 gene regulation in ECs
in response to starvation and angiogenic growth factor stimulation.
Cells are microvascular ECs isolated from lung. The first sample
(70% con) is sub-confluent, rapidly proliferating cells in rich
medium. The second sample (90% con) is still proliferating in rich
medium, but proliferation is slowing as the cells approach
confluence. The third sample (18h starve) represents cells
incubated for 18 h in the absence of serum and angiogenic growth
factors. These are starved cells, and the remaining 8 samples start
from starved cells, which then go on to further treatment as
follows: bFGF (basic fibroblast growth factor) and VEGF (vascular
endothelial growth factor) are used at 5 nM. CM indicates both, NGF
indicates "No growth factor"--neither is added and the cells
continue to starve. The middle group of samples is cells treated
with growth factors (or not) for 2 hours after the 18 h starve. The
right group are cells treated for 18 h after the 18 h starve.
[0508] Down-regulation of 279 mRNA is detected as cells become
confluent (70% vs. 90%) or with starvation (70% and/or 90% vs. 18h
starve), and upregulation of the mRNA at 2 h or 18 h in response to
both growth factors (CM) and/or a single growth factor (FIG. 7).
These results support an angiogenic function of 279 polypeptide.
Id1 is used as a control gene. It is required for angiogenesis in
vivo (Lyden, D. et. al. (1999) Nature 401(6754):670-7).
[0509] Equivalents
[0510] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims
Sequence CWU 1
1
3 1 1089 DNA Homo sapien 1 atgggcaacc acacgtggga gggctgccac
gtggactcgc gcgtggacca cctctttccg 60 ccatccctct acatctttgt
catcggcgtg gggctgccca ccaactgcct ggctctgtgg 120 gcggcctacc
gccaggtgca acagcgcaac gagctgggcg tctacctgat gaacctcagc 180
atcgccgacc tgctgtacat ctgcacgctg ccgctgtggg tggactactt cctgcaccac
240 gacaactgga tccacggccc cgggtcctgc aagctctttg ggttcatctt
ctacaccaat 300 atctacatca gcatcgcctt cctgtgctgc atctcggtgg
accgctacct ggctgtggcc 360 cacccactcc gcttcgcccg cctgcgccgc
gtcaagaccg ccgtggccgt gagctccgtg 420 gtctgggcca cggagctggg
cgccaactcg gcgcccctgt tccatgacga gctcttccga 480 gaccgctaca
accacacctt ctgctttgag aagttcccca tggaaggctg ggtggcctgg 540
atgaacctct atcgggtgtt cgtgggcttc ctcttcccgt gggcgctcat gctgctgtcg
600 taccggggca tcctgcgggc cgtgcggggc agcgtgtcca ccgagcgcca
ggagaaggcc 660 aagatcaagc ggctggccct cagcctcatc gccatcgtgc
tggtctgctt tgcgccctat 720 cacgtgctct tgctgtcccg cagcgccatc
tacctgggcc gcccctggga ctgcggcttc 780 gaggagcgcg tcttttctgc
ataccacagc tcactggctt tcaccagcct caactgtgtg 840 gcggacccca
tcctctactg cctggtcaac gagggcgccc gcagcgatgt ggccaaggcc 900
ctgcacaacc tgctccgctt tctggccagc gacaagcccc aggagatggc caatgcctcg
960 ctcaccctgg agaccccact cacctccaag aggaacagca cagccaaagc
catgactggc 1020 agctgggcgg ccactccgcc ctcccagggg gaccaggtgc
agctgaagat gctgccgcca 1080 gcacaatga 1089 2 362 PRT Homo sapien 2
Met Gly Asn His Thr Trp Glu Gly Cys His Val Asp Ser Arg Val Asp 1 5
10 15 His Leu Phe Pro Pro Ser Leu Tyr Ile Phe Val Ile Gly Val Gly
Leu 20 25 30 Pro Thr Asn Cys Leu Ala Leu Trp Ala Ala Tyr Arg Gln
Val Gln Gln 35 40 45 Arg Asn Glu Leu Gly Val Tyr Leu Met Asn Leu
Ser Ile Ala Asp Leu 50 55 60 Leu Tyr Ile Cys Thr Leu Pro Leu Trp
Val Asp Tyr Phe Leu His His 65 70 75 80 Asp Asn Trp Ile His Gly Pro
Gly Ser Cys Lys Leu Phe Gly Phe Ile 85 90 95 Phe Tyr Thr Asn Ile
Tyr Ile Ser Ile Ala Phe Leu Cys Cys Ile Ser 100 105 110 Val Asp Arg
Tyr Leu Ala Val Ala His Pro Leu Arg Phe Ala Arg Leu 115 120 125 Arg
Arg Val Lys Thr Ala Val Ala Val Ser Ser Val Val Trp Ala Thr 130 135
140 Glu Leu Gly Ala Asn Ser Ala Pro Leu Phe His Asp Glu Leu Phe Arg
145 150 155 160 Asp Arg Tyr Asn His Thr Phe Cys Phe Glu Lys Phe Pro
Met Glu Gly 165 170 175 Trp Val Ala Trp Met Asn Leu Tyr Arg Val Phe
Val Gly Phe Leu Phe 180 185 190 Pro Trp Ala Leu Met Leu Leu Ser Tyr
Arg Gly Ile Leu Arg Ala Val 195 200 205 Arg Gly Ser Val Ser Thr Glu
Arg Gln Glu Lys Ala Lys Ile Lys Arg 210 215 220 Leu Ala Leu Ser Leu
Ile Ala Ile Val Leu Val Cys Phe Ala Pro Tyr 225 230 235 240 His Val
Leu Leu Leu Ser Arg Ser Ala Ile Tyr Leu Gly Arg Pro Trp 245 250 255
Asp Cys Gly Phe Glu Glu Arg Val Phe Ser Ala Tyr His Ser Ser Leu 260
265 270 Ala Phe Thr Ser Leu Asn Cys Val Ala Asp Pro Ile Leu Tyr Cys
Leu 275 280 285 Val Asn Glu Gly Ala Arg Ser Asp Val Ala Lys Ala Leu
His Asn Leu 290 295 300 Leu Arg Phe Leu Ala Ser Asp Lys Pro Gln Glu
Met Ala Asn Ala Ser 305 310 315 320 Leu Thr Leu Glu Thr Pro Leu Thr
Ser Lys Arg Asn Ser Thr Ala Lys 325 330 335 Ala Met Thr Gly Ser Trp
Ala Ala Thr Pro Pro Ser Gln Gly Asp Gln 340 345 350 Val Gln Leu Lys
Met Leu Pro Pro Ala Gln 355 360 3 275 PRT Artificial Sequence
Consensus sequence 3 Gly Asn Leu Leu Val Ile Leu Val Ile Leu Arg
Thr Lys Lys Leu Arg 1 5 10 15 Thr Pro Thr Asn Ile Phe Ile Leu Asn
Leu Ala Val Ala Asp Leu Leu 20 25 30 Phe Leu Leu Thr Leu Pro Pro
Trp Ala Leu Tyr Tyr Leu Val Gly Gly 35 40 45 Ser Glu Asp Trp Pro
Phe Gly Ser Ala Leu Cys Lys Leu Val Thr Ala 50 55 60 Leu Asp Val
Val Asn Met Tyr Ala Ser Ile Leu Leu Leu Thr Ala Ile 65 70 75 80 Ser
Ile Asp Arg Tyr Leu Ala Ile Val His Pro Leu Arg Tyr Arg Arg 85 90
95 Arg Arg Thr Ser Pro Arg Arg Ala Lys Val Val Ile Leu Leu Val Trp
100 105 110 Val Leu Ala Leu Leu Leu Ser Leu Pro Pro Leu Leu Phe Ser
Trp Val 115 120 125 Lys Thr Val Glu Glu Gly Asn Gly Thr Leu Asn Val
Asn Val Thr Val 130 135 140 Cys Leu Ile Asp Phe Pro Glu Glu Ser Thr
Ala Ser Val Ser Thr Trp 145 150 155 160 Leu Val Ser Tyr Val Leu Leu
Ser Thr Leu Val Gly Phe Leu Leu Pro 165 170 175 Leu Leu Val Ile Leu
Val Cys Tyr Thr Arg Ile Leu Arg Thr Leu Arg 180 185 190 Lys Arg Ala
Arg Lys Gly Ala Ser Lys Lys Arg Ser Ser Lys Glu Arg 195 200 205 Lys
Ala Ala Lys Thr Leu Leu Val Val Val Val Val Phe Val Leu Cys 210 215
220 Trp Leu Pro Tyr Phe Ile Val Leu Leu Asp Thr Leu Cys Xaa Leu Ser
225 230 235 240 Ile Ile Met Ser Ser Thr Cys Glu Leu Glu Arg Val Leu
Pro Thr Ala 245 250 255 Leu Leu Val Thr Leu Trp Leu Ala Tyr Val Asn
Ser Cys Leu Asn Pro 260 265 270 Ile Ile Tyr 275
* * * * *
References